Genetic markers for risk management of atrial fibrillation, atrial flutter, and stroke

ABSTRACT

The invention relates to procedure and methods of determining a susceptibility to cardiac arrhythmia, including Atrial Fibrillation, Atrial Flutter and Stroke, by assessing the presence or absence of alleles at polymorphic markers found to be associated with Atrial Fibrillation, Atrial Flutter and Stroke. The invention further relates to kits encompassing reagents for assessing such markers, and diagnostic methods, uses and procedures for utilizing such susceptibility markers.

BACKGROUND OF THE INVENTION

Cardiac arrhythmia is a group of medical conditions, in which theelectrical activity of the heart is irregular, or is slower or fasterthan normal. Some arrhythmias are life-threatening, and can causecardiac arrest or sudden death. Others cause, or predispose to, otheraggravating symptoms or disease, including stroke. Fibrillation is aserious form of arrhythmia, in which the heart muscle presents withirregular or quivering motion due to lack of unity in the function ofcontractile cells. Fibrillation can affect the atrium (AtrialFibrillation (AF) or Atrial Flutter (AFI)), or the ventricle(Ventricular Fibrillation (VF)).

Atrial fibrillation (AF) is an abnormal heart rhythm (cardiacarrhythmia) which involves the two small, upper heart chambers (theatria). Heart beats in a normal heart begin after electricity generatedin the atria by the sinoatrial node spreads through the heart and causescontraction of the heart muscle and pumping of blood. In AF, the regularelectrical impulses of the sinoatrial node are replaced by disorganized,rapid electrical impulses which result in irregular heart beats.

Atrial fibrillation is the most common cardiac arrhythmia. The risk ofdeveloping atrial fibrillation increases with age—AF affects fourpercent of individuals in their 80s. An individual may spontaneouslyalternate between AF and a normal rhythm (paroxysmal atrialfibrillation) or may continue with AF as the dominant cardiac rhythmwithout reversion to the normal rhythm (chronic atrial fibrillation).Atrial fibrillation is often asymptomatic, but may result in symptoms ofpalpitations, fainting, chest pain, or even heart failure. Thesesymptoms are especially common when atrial fibrillation results in aheart rate which is either too fast or too slow. In addition, theerratic motion of the atria leads to blood stagnation (stasis) whichincreases the risk of blood clots that may travel from the heart to thebrain and other areas. Thus, AF is an important risk factor for stroke,the most feared complication of atrial fibrillation.

The symptoms of atrial fibrillation may be treated with medicationswhich slow the heart rate. Several medications as well as electricalcardioversion may be used to convert AF to a normal heart rhythm.Surgical and catheter-based therapies may also be used to prevent atrialfibrillation in certain individuals. People with AF are often givenblood thinners such as warfarin to protect them from strokes.

Any patient with 2 or more identified episodes of atrial fibrillation issaid to have recurrent atrial fibrillation. This is further classifiedinto paroxysmal and persistent based on when the episode terminateswithout therapy. Atrial fibrillation is said to be paroxysmal when itterminates spontaneously within 7 days, most commonly within 24 hours.Persistent or chronic atrial fibrillation is AF established for morethan seven days. Differentiation of paroxysmal from chronic orestablished AF is based on the history of recurrent episodes and theduration of the current episode of AF (Levy S., J CardiovascElectrophysiol. 8 Suppl, S78-82 (1998)).

Lone atrial fibrillation (LAF) is defined as atrial fibrillation in theabsence of clinical or echocardiographic findings of cardiopulmonarydisease.

Atrial fibrillation is usually accompanied by symptoms related to eitherthe rapid heart rate or embolization. Rapid and irregular heart ratesmay be perceived as palpitations, exercise intolerance, and occasionallyproduce angina and congestive symptoms of shortness of breath or edema.Sometimes the arrhythmia will be identified with the onset of a strokeor a transient ischemic attack (TIA). It is not uncommon to identifyatrial fibrillation on a routine physical examination orelectrocardiogram (ECG/EKG), as it may be asymptomatic in some cases.Paroxysmal atrial fibrillation is the episodic occurrence of thearrhythmia and may be difficult to diagnose. Episodes may occur withsleep or with exercise, and their episodic nature may require prolongedECG monitoring (e.g. a Holter monitor) for diagnosis.

Atrial fibrillation is diagnosed on an electrocardiogram, aninvestigation performed routinely whenever irregular heart beat issuspected. Characteristic findings include absence of P waves,unorganized electrical activity in their place and irregularity of R-Rinterval due to irregular conduction of impulses to the ventricles. Ifparoxysmal AF is suspected, episodes may be documented with the use ofHolter monitoring (continuous ECG recording for 24 hours or longer).

While many cases of AF have no definite cause, it may be the result ofvarious other problems (see below). Hence, renal function andelectrolytes are routinely determined, as well as thyroid-stimulatinghormone and a blood count. A chest X-ray is generally performed. Inacute-onset AF associated with chest pain, cardiac troponins or othermarkers of damage to the heart muscle may be ordered. Coagulationstudies (INR/aPTT) are usually performed, as anticoagulant medicationmay be commenced. A transesophageal echocardiogram may be indicated toidentify any intracardiac thrombus (Fuster V., et al., Circulation.;104, 2118-2150 (2001)).

Atrial Flutter (AFI) is characterized by an abnormal fast heart rhythmin the atria. Patients who present with atrial flutter commonly alsoexperience Atrial Fibrillation and vice versa (Waldo, A., ProgrCardiovasc Disease, 48:41-56 (2005)). Mechanistically and biologically,AF and AFI are thus likely to be highly related.

AF (and AFI) is linked to several cardiac causes, but may occur inotherwise normal hearts. Known associations include: High bloodpressure, Mitral stenosis (e.g. due to rheumatic heart disease or mitralvalve prolapse), Mitral regurgitation, Heart surgery, Coronary arterydisease, Hypertrophic cardiomyopathy, Excessive alcohol consumption(“binge drinking” or “holiday heart”), Hyperthyroidism, Hyperstimulationof the vagus nerve, usually by having large meals (“binge eating”), Lungpathology (such as pneumonia, lung cancer, pulmonary embolism,Sarcoidosis), Pericarditis, Intense emotional turmoil, and Congenitalheart disease.

The normal electrical conduction system of the heart allows the impulsethat is generated by the sinoatrial node (SA node) of the heart to bepropagated to and stimulate the myocardium (muscle of the heart). Whenthe myocardium is stimulated, it contracts. It is the orderedstimulation of the myocardium that allows efficient contraction of theheart, thereby allowing blood to be pumped to the body. In atrialfibrillation, the regular impulses produced by the sinus node to providerhythmic contraction of the heart are overwhelmed by the rapid randomlygenerated discharges produced by larger areas of atrial tissue. Anorganized electrical impulse in the atrium produces atrial contraction;the lack of such an impulse, as in atrial fibrillation, producesstagnant blood flow, especially in the atrial appendage and predisposesto clotting. The dislodgement of a clot from the atrium results in anembolus, and the damage produced is related to where the circulationtakes it. An embolus to the brain produces the most feared complicationof atrial fibrillation, stroke, while an embolus may also lodge in themesenteric circulation (the circulation supplying the abdominal organs)or digit, producing organ-specific damage.

Treatment of atrial fibrillation is directed by two main objectives: (i)prevent temporary circulatory instability; (ii) prevent stroke. The mostcommon methods for achieving the former includes rate and rhythmcontrol, while anticoagulation is usually the desired method for thelatter (Prystowsky E. N., Am J Cardiol.; 85, 3D-11D (2000); van WalravenC, et al., Jama. 288, 2441-2448 (2002)). Common methods for ratecontrol, i.e. for reducing heart rate to normal, include beta blockers(e.g., metotprolol), cardiac glycosides (e.g., digoxin) and calciumchannel blockers (e.g., verapamil). All these medications work byslowing down the generation of pulses from the atria, and the conductionfrom the atria to the ventricles. Other drugs commonly used includequinidine, flecainide, propafenone, disopyramide, sotalol andamiodarone. Rhythm control can be achieved by electrical cardioversion,i.e. by applying DC electrical shock, or by chemical cardioversion,using drugs such as amiodarione, propafenone and flecainide.

Preventive measures for stroke include anticoagulants. Representativeexamples of anticoagulant agents are Dalteparin (e.g., Fragmin),Danaparoid (e.g., Orgaran), Enoxaparin (e.g., Lovenox), Heparin(various), Tinzaparin (e.g., Innohep), Warfarin (e.g., Coumadin). Somepatients with lone atrial fibrillation are sometimes treated withaspirin or clopidogrel. There is evidence that aspirin and clopidogrelare effective when used together, but the combination is still inferiorto warfarin (Connolly S., et al. Lancet.; 367, 1903-1912 (2006)).(2) Thenew anticoagulant ximelagatran has been shown to prevent stroke withequal efficacy as warfarin, without the difficult monitoring processassociated with warfarin and with possibly fewer adverse haemorrhagicevents. Unfortunately, ximegalatran and other similar anticoagulantdrugs (commonly referred to as direct thrombin inhibitors), have yet tobe widely licensed.

Determining who should and should not receive anti-coagulation withwarfarin is not straightforward. The CHADS2 score is the best validatedmethod of determining risk of stroke (and therefore who should beanticoagulated). The UK NICE guidelines have instead opted for analgorithm approach. The underlying problem is that if a patient has ayearly risk of stroke that is less than 2%, then the risks associatedwith taking warfarin outweigh the risk of getting a stroke (Gage B. F.et al. Stroke 29, 1083-1091 (1998))

Atrial fibrillation can sometimes be controlled with treatment. Thenatural tendency of atrial fibrillation, however, is to become a chroniccondition. Chronic AF leads to an increased risk of death. Patients withatrial fibrillation are at significantly increased chance of stroke.

Atrial fibrillation is common among older adults. In developedcountries, the number of patients with atrial fibrillation is likely toincrease during the next 50 years, due to the growing proportion ofelderly individuals (Go A. S. et al., Jama., 285, 2370-2375 (2001))(3).In the Framingham study the lifetime risk for development of AF is 1 in4 for men and women 40 years of age and older. Lifetime risks for AF arehigh (1 in 6). According to data from the National Hospital DischargeSurvey (1996-2001) on cases that included AF as a primary dischargediagnosis found that 45% of the patients are male, and that the mean agefor men was 66.8 years and 74.6 for women. The racial breakdown foradmissions was found to be 71.2% white, 5.6% black, 2% other races, and20% not specified. Furthermore, African American patients were, onaverage, much younger than other races. The incidence in men ranged from20.58/100,000 persons per year for patients ages 15-44 years to1203/100,000 persons per years for those ages 85 and older. From1996-2001, hospitalizations with AF as the first listed diagnosis,increased by 34%.

Stroke is a common and serious disease. Each year in the United Statesmore than 600,000 individuals suffer a stroke and more than 160,000 diefrom stroke-related causes (Sacco, R. L. et al., Stroke 28, 1507-17(1997)). Furthermore, over 300,000 individuals present with TransientIschemic Attack, a mild form of stroke, every year in the US. In westerncountries stroke is the leading cause of severe disability and the thirdleading cause of death (Bonita, R., Lancet 339, 342-4 (1992)). Thelifetime risk of those who reach the age of 40 exceeds 10%.

The clinical phenotype of stroke is complex but is broadly divided intoischemic (accounting for 80-90%) and hemorrhagic stroke (10-20%)(Caplan, L. R. Caplan's Stroke: A Clinical Approach, 1-556(Butterworth-Heinemann, 2000)). Ischemic stroke is further subdividedinto large vessel occlusive disease (referred to here as carotidstroke), usually due to atherosclerotic involvement of the common andinternal carotid arteries, small vessel occlusive disease, thought to bea non-atherosclerotic narrowing of small end-arteries within the brain,and cardiogenic stroke due to blood clots arising from the heart usuallyon the background of atrial fibrillation or ischemic (atherosclerotic)heart disease (Adams, H. P., Jr. et al., Stroke 24, 35-41 (1993)).Therefore, it appears that stroke is not one disease but a heterogeneousgroup of disorders reflecting differences in the pathogenic mechanisms(Alberts, M. J. Genetics of Cerebrovascular Disease, 386 (FuturaPublishing Company, Inc., New York, 1999); Hassan, A. & Markus, H. S.Brain 123, 1784-812 (2000)). However, all forms of stroke share riskfactors such as hypertension, diabetes, hyperlipidemia, and smoking(Sacco, R. L. et al., Stroke 28, 1507-17 (1997); Leys, D. et al., J.Neurol. 249, 507-17 (2002)). Family history of stroke is also anindependent risk factor suggesting the existence of genetic factors thatmay interact with environmental factors (Hassan, A. & Markus, H. S.Brain 123, 1784-812 (2000); Brass, L. M. & Alberts, M. J. BaillieresClin. Neurol. 4, 221-45 (1995)).

The genetic determinants of the common forms of stroke are still largelyunknown. There are examples of mutations in specific genes that causerare Mendelian forms of stroke such as the Notch3 gene in CADASIL(cerebral autosomal dominant arteriopathy with subcortical infarctionsand leukoencephalopathy) (Tournier-Lasserve, E. et al., Nat. Genet. 3,256-9 (1993); Joutel, A. et al., Nature 383, 707-10 (1996)), Cystatin Cin the Icelandic type of hereditary cerebral hemorrhage with amyloidosis(Palsdottir, A. et al., Lancet 2, 603-4 (1988)), APP in the Dutch typeof hereditary cerebral hemorrhage (Levy, E. et al., Science 248, 1124-6(1990)) and the KRIT1 gene in patients with hereditary cavernous angioma(Gunel, M. et al., Proc. Natl. Acad. Sci. USA 92, 6620-4 (1995); Sahoo,T. et al., Hum. Mol. Genet. 8, 2325-33 (1999)). None of these rare formsof stroke occur on the background of atherosclerosis, and therefore, thecorresponding genes are not likely to play roles in the common forms ofstroke which most often occur with atherosclerosis.

It is very important for the health care system to develop strategies toprevent stroke. Once a stroke happens, irreversible cell death occurs ina significant portion of the brain supplied by the blood vessel affectedby the stroke. Unfortunately, the neurons that die cannot be revived orreplaced from a stem cell population. Therefore, there is a need toprevent strokes from happening in the first place. Although we alreadyknow of certain clinical risk factors that increase stroke risk (listedabove), there is an unmet medical need to define the genetic factorsinvolved in stroke to more precisely define stroke risk. Further, ifpredisposing alleles are common in the general population and thespecificity of predicting a disease based on their presence is low,additional loci such as protective loci are needed for meaningfulprediction of disposition of the disease state. There is also a greatneed for therapeutic agents for preventing the first stroke or furtherstrokes in individuals who have suffered a previous stroke or transientischemic attack.

AF is an independent risk factor for stroke, increasing risk about5-fold. The risk for stroke attributable to AF increases with age. AF isresponsible for about 15-20% of all strokes. AF is also an independentrisk factor for stroke recurrence and stroke severity. A recent reportshowed people who had AF and were not treated with anticoagulants had a2.1-fold increase in risk for recurrent stroke and a 2.4 fold increasein risk for recurrent severe stroke. People who have stroke caused by AFhave been reported as 2.23 times more likely to be bedridden compared tothose who have strokes from other causes.

There is a need for an understanding of the susceptibility factorsleading to increased predisposition for AF and stroke. Identification ofat-risk variants for AF can, for example, be useful for assessing whichindividuals are at particularly high risk for AF and subsequent stroke.Furthermore, preventive treatment can be administered to individualssuffering from AF and who are carriers of at-risk susceptibilityvariants for AF and/or stroke. Finally, identification of at-riskvariants for AF and/or stroke can lead to the identification of newtargets for drug therapy, as well as the development of noveltherapeutic measures.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that certain geneticmarkers have been shown to be associated with cardiac arrhythmia, inparticular atrial fibrillation and atrial flutter, and stroke. Thisdiscovery can be utilized in a variety of methods, procedures,apparatus, media and kits, as described herein, relating to methods andprocedures of diagnosis and/or determination of a susceptibility,methods of genotyping associated variants, methods of predictingresponse to therapeutic agents, methods of predicting prognosis, methodsof monitoring progress of treatment, and systems and kits for use insuch methods.

One aspect of the invention relates to a method of determining asusceptibility to cardiac arrhythmia or stroke in a human individual,the method comprising determining the presence or absence of at leastone allele of at least one polymorphic marker in a nucleic acid samplefrom the individual, wherein the at least one polymorphic marker isselected from the polymorphic markers set forth in Table 5, and markersin linkage disequilibrium therewith, wherein determination of thepresence or absence of the at least one allele is indicative of asusceptibility to cardiac arrhythmia or stroke in the individual. In oneembodiment, the at least one polymorphic marker is located within the LDblock C04, set forth in SEQ ID NO:50 herein. In another embodiment, theat least one polymorphic marker is selected from the markers set forthin Table 9, and markers in linkage disequilibrium therewith. In oneembodiment, the at least one marker is selected from marker rs2220427(SEQ ID NO:1) and marker rs10033464 (SEQ ID NO:41), and markers inlinkage disequilibrium therewith. In another embodiment, the at leastone polymorphic marker is selected from the markers set forth in Table19. In one embodiment, the method further comprises a step of assessingat least one haplotype comprising at least two polymorphic markers inthe individual.

In another aspect, the invention relates to a method of determining asusceptibility to cardiac arrhythmia or stroke in a human individual,comprising determining whether at least one at-risk allele in at leastone polymorphic marker is present in a genotype dataset derived from theindividual, wherein the at least one polymorphic marker is selected fromthe markers set forth in Table 5, and markers in linkage disequilibriumtherewith, and wherein determination of the presence of the at least oneat-risk allele is indicative of increased susceptibility to cardiacarrhythmia or stroke in the individual.

The genotype dataset comprises in one embodiment information aboutmarker identity, and the allelic status of the individual for the atleast one polymorphic marker, i.e. information about the identity of thetwo alleles carried by the individual for the marker and/or informationabout whether an individual is a carrier of a particular at-risk allelefor the at least one polymorphic marker. The genotype dataset maycomprise allelic information about one or more marker, including two ormore markers, three or more markers, five or more markers, one hundredor more markers, etc. In some embodiments, the genotype datasetcomprises genotype information from a whole-genome assessment of theindividual including hundreds of thousands of markers, or even onemillion or more markers.

The invention, in another aspect, relates to a procedure comprising astep of analyzing a nucleic acid from a human individual to determinethe presence or absence of at least one allele of at least onepolymorphic marker or haplotype associated with the genomic sequencewith sequence as set forth in SEQ ID NO:50; and a step of determiningthe status of a genetic indicator of cardiac arrhythmia or stroke in theindividual from the presence or absence of the at least one marker orhaplotype. Thus the genotype and/or haplotype status of the individualis used as in indicator of cardiac arrhythmia, including atrialfibrillation and atrial flutter, as well as stroke, in the individual.

The invention also relates to a method of assessing a susceptibility tocardiac arrhythmia or stroke in a human individual, comprising screeninga nucleic acid from the individual for at least one polymorphic markeror haplotype in SEQ ID NO:50 that correlates with increased occurrenceof cardiac arrhythmia or stroke in a human population; whereindetermination of the presence of an at-risk marker allele in the atleast one polymorphism or an at-risk haplotype in the nucleic acididentifies the individual as having elevated susceptibility to cardiacarrhythmia and/or stroke, and wherein the absence of the at least oneat-risk marker allele or at-risk haplotype in the nucleic acididentifies the individual as not having the elevated susceptibility.

The procedure or methods of the invention in one embodiment entail atleast one polymorphic marker or haplotype comprising a contiguousnucleic acid fragment of LD block C04 as set forth in SEQ ID NO:50, orthe complement thereof, wherein the fragment is less than 500nucleotides in size and specifically hybridizes to a complimentarysegment of LD block C04. In one embodiment, the fragment is more than 15nucleotides and less than 400 nucleotides in size, and wherein thefragment specifically hybridizes to a complimentary segment of LD blockC04 as set forth in SEQ ID NO:50.

In alternative embodiments, the susceptibility conferred by thepolymorphic markers or haplotypes is decreased susceptibility, i.e. themarkers and haplotypes of the invention confer decreased risk of anindividual develops cardiac arrhythmia, including atrial fibrillationand atrial flutter, and/or stroke. In one such embodiment, the decreasedsusceptibility is characterized by an odds ratio (OR) or relative risk(RR) of less than 0.8. In another embodiment, the decreasedsusceptibility is characterized by an odds ratio (OR) of less than 0.7.In another embodiment, the decreased susceptibility is characterized byan OR or RR of less than 0.6. In another embodiment, the decreasedsusceptibility is characterized by OR or RR of less than 0.5. Otherembodiments relate to other values for OR or RR including values of 0.9,0.85, 0.75, 0.65, 0.55, etc.

Another aspect of the invention relates to a method of identification ofa marker for use in assessing susceptibility to symptoms associated withcardiac arrhythmia and/or stroke in a human individual, the methodcomprising at least one polymorphic marker within SEQ ID NO:50, or atleast one polymorphic marker in linkage disequilibrium with at least onemarker within SEQ ID NO:50, determining the genotype status of a sampleof individuals diagnosed with cardiac arrhythmia and/or stroke and thegenotype status of a sample of control individuals, wherein asignificant difference in frequency of at least one allele in at leastone polymorphism in individuals diagnosed with cardiac arrhythmia and/orstroke as compared with the frequency of the at least one allele in thecontrol sample is indicative of the at least one polymorphism beinguseful for assessing susceptibility to cardiac arrhythmia and/or stroke.In one embodiment, an increase in frequency of the at least one allelein the at least one polymorphism in individuals diagnosed with cardiacarrhythmia and/or stroke, as compared with the frequency of the at leastone allele in the control sample, is indicative of the at least onepolymorphism being useful for assessing increased susceptibility tocardiac arrhythmia. In another embodiment, a decrease in frequency ofthe at least one allele in the at least one polymorphism in individualsdiagnosed with cardiac arrhythmia and/or stroke, as compared with thefrequency of the at least one allele in the control sample, isindicative of the at least one polymorphism being useful for assessingdecreased susceptibility to, or protection against, cardiac arrhythmiaand/or stroke. In preferred embodiments, the significant difference infrequency is characterized by a statistical measure. In one embodiment,the statistical measure is a P-value. In particular embodiments, asignificant P-value is less than 0.05, less than 0.01, less than 0.001,less than 0.0001, less than 0.00001, less than 0.000001, less than0.0000001 or less than 0.00000001. In other embodiments, the significantdifference is characterized by an odds ratio (OR) or relative risk (RR)with particular confidence interval (CE) values.

In another aspect, the invention relates to a method of genotyping anucleic acid sample obtained from a human individual, comprisingdetermining the presence or absence of at least one allele of at leastone polymorphic marker predictive of increased risk of cardiacarrhythmia and/or stroke in the sample, wherein the at least one markeris selected from the markers set forth in Table 5, and markers inlinkage disequilibrium therewith, and wherein determination of thepresence or absence of the at least one allele of the at least onepolymorphic marker is predictive of increased risk of cardiac arrhythmiaand/or stroke in the individual. In one embodiment, genotyping isperformed using a process selected from allele-specific probehybridization, allele-specific primer extension, allele-specificamplification, nucleic acid sequencing, 5′-exonuclease digestion,molecular beacon assay, oligonucleotide ligation assay, size analysis,and single-stranded conformation analysis. In a preferred embodiment,the process comprises allele-specific probe hybridization. The processof genotyping preferably comprises amplifying a segment of a nucleicacid that comprises the at least one polymorphic marker, by PolymeraseChain Reaction (PCR), using a nucleotide primer pair flanking the atleast one polymorphic marker. In a preferred method of genotyping, thefollowing steps are performed:

-   -   1. contacting copies of the nucleic acid with a detection        oligonucleotide probe and an enhancer oligonucleotide probe        under conditions for specific hybridization of the        oligonucleotide probe with the nucleic acid; wherein        -   a) the detection oligonucleotide probe is from 5-100            nucleotides in length and specifically hybridizes to a first            segment of the nucleic acid whose nucleotide sequence is            given by SEQ ID NO:50 that comprises at least one            polymorphic site;        -   b) the detection oligonucleotide probe comprises a            detectable label at its 3′ terminus and a quenching moiety            at its 5′ terminus;        -   c) the enhancer oligonucleotide is from 5-100 nucleotides in            length and is complementary to a second segment of the            nucleotide sequence that is 5′ relative to the            oligonucleotide probe, such that the enhancer            oligonucleotide is located 3′ relative to the detection            oligonucleotide probe when both oligonucleotides are            hybridized to the nucleic acid; and        -   d) a single base gap exists between the first segment and            the second segment, such that when the oligonucleotide probe            and the enhancer oligonucleotide probe are both hybridized            to the nucleic acid, a single base gap exists between the            oligonucleotides;    -   2. treating the nucleic acid with an endonuclease that will        cleave the detectable label from the 3′ terminus of the        detection probe to release free detectable label when the        detection probe is hybridized to the nucleic acid; and        measuring free detectable label, wherein the presence of the        free detectable label indicates that the detection probe        specifically hybridizes to the first segment of the nucleic        acid, and indicates the sequence of the polymorphic site as the        complement of the detection probe.

A further aspect of the invention relates to a method of determining asusceptibility to cardiac arrhythmia or stroke in a human individual,the method comprising determining the identity of at least one allele ofat least one polymorphic marker in a nucleic acid sample obtained fromthe individual, wherein the at least one marker is selected from thegroup of markers associated with the PITX2 gene, wherein the presence ofthe at least one allele is indicative of a susceptibility to cardiacarrhythmia or stroke in the individual.

Some embodiments of the invention relate to a further step of assessingat least one additional biomarker for atrial fibrillation, atrialflutter or stroke, wherein combining the genetic information from themarkers provides risk assessment for atrial fibrillation, atrial flutteror stroke. In some of these embodiments, the biomarker is a geneticmarker or haplotype, i.e. genetic risk factors shown to be, orcontemplated to be, related to increased or decreased risk of atrialfibrillation, atrial flutter or stroke. In other embodiments thebiomarker is a protein biomarker. The protein biomarker is in someembodiments selected from fibrin D-dimer, prothrombin activationfragment 1.2 (F1.2), thrombin-antithrombin III complexes (TAT),fibrinopeptide A (FPA), lipoprotein-associated phospholipase A2(Ip-PLA2), beta-thromboglobulin, platelet factor 4, P-selectin, vonWillebrand Factor, pro-natriuretic peptide (BNP), matrixmetalloproteinase-9 (MMP-9), PARK7, nucleoside diphosphate kinase(NDKA), tau, neuron-specific enolase, B-type neurotrophic growth factor,astroglial protein S-100b, glial fibrillary acidic protein, C-reactiveprotein, seum amyloid A, marix metalloproteinase-9, vascular andintracellular cell adhesion molecules, tumor necrosis factor alpha, andinterleukins, including interleukin-1, -6, and -8). In one embodiment,the at least one biomarker includes progenitor cells. In particularembodiments, more than one biomarker is determined. In a preferredembodiment, the biomarker is measured in plasma from the individual.Other embodiments further relate to combining non-genetic information tomake risk assessment, diagnosis, or prognosis of atrial fibrillation,atrial flutter or stroke in the individual. The non-genetic informationcan comprise age, age at onset of disease, gender, ethnicity, previousdisease diagnosis, e.g., diagnosis of cardiag arrhythmia (e.g., atrialfibrillation) and stroke, medical history of the individual, familyhistory of disease, biochemical measurements, and clinical measurements(e.g., blood pressure, serum lipid levels). Analysis of such combinedinformation from various genetic markers, or genetic markers plusnon-genetic markers is possible by methods known to those skilled in theart. In one embodiment, analysis is performed calculating overall riskby logistic regression.

The invention further relates to a method of diagnosing increasedsusceptibility of stroke in a human individual, comprising the steps of(a) determining whether the individual has experienced symptomsassociated with Atrial Fibrillation, Atrial Flutter or a TransientIschemic Attack; (b) determining whether a nucleic acid sample from theindividual comprises at least one copy of an at-risk allele of at leastone polymorphic marker selected from the markers set forth in Table 5,and markers in linkage disequilibrium therewith; wherein the presence ofsymptoms associated with Atrial Fibrillation, Atrial Flutter and/orTransient Ischemic Attack and the presence of the at least one copy ofthe at-risk allele is indicative of increased susceptibility of stroke.

The invention in a further aspect relates to a method of assessing anindividual for probability of response to a therapeutic agent forpreventing and/or ameliorating symptoms associated with cardiacarrhythmia and/or stroke, comprising: determining the presence orabsence of at least one allele of at least one polymorphic marker in anucleic acid sample obtained from the individual, wherein the at leastone polymorphic marker is selected from the markers set forth in Table9, and markers in linkage disequilibrium therewith, whereindetermination of the presence of the at least one allele of the at leastone marker is indicative of a probability of a positive response to thetherapeutic agent for cardiac arrhythmia and/or stroke.

In one embodiment, the therapeutic agent is an anticoagulant, ananti-arrhythmic agent, a hear rate control agent, a cardioversion agent,or a heart rhythm control agent. In another embodiment, the therapeuticagent is selected from warfarin, heparin, low molecular weight heparins,factor Xa inhibitors, and thrombin inhibitors, sodium channel blockers,beta blockers, potassium channel blockers, and calcium channel blockers.

In another embodiment, the therapeutic agent is selected from warfaring,ximelagatran, heparin, enoxaparin, dalteparin, tinzaparin, ardeparin,nadroparin, reviparin, fondaparinux, idraparinux, lepirudin,bivalirudin, argatroban, danaparoid, disopyramide, moricizine,procainamide, quinidine, lidocaine, mexiletine, tocainide, phenytoin,encainide, flecainide, propafenone, ajmaline, cibenzoline, detajmium,esmolol, propranolol, metoprolol, alprenolol, atenolol, carvedilol,bisoprolol, acebutolol, nadolol, pindololol, labetalol, oxprenotol,penbutolol, timolol, betaxolol, cartelol, sotalol, levobunolol,amiodarone, azimilide, bretylium, dofetilide, tedisamil, ibutilide,sematilide, N-acetyl procainamide, nifekalant hydrochloride,vernakalant, ambasilide, verpamil, mibefradil, diltiazem, digoxin,adenosine, ibutilide, amiodarone, procainamide, profafenone andflecainide.

Yet another aspect of the invention relates to a method of predictingprognosis of an individual diagnosed with, cardiac arrhythmia and/orstroke, the method comprising determining the presence or absence of atleast one allele of at least one polymorphic marker in a nucleic acidsample obtained from the individual, wherein the at least onepolymorphic marker is selected from the markers set forth in Table 9,and markers in linkage disequilibrium therewith, wherein determinationof the presence of the at least one allele is indicative of a worseprognosis of the cardiac arrhythmia and/or stroke in the individual.

Methods of monitoring progress of a treatment of an individualundergoing treatment for cardiac arrhythmia and/or stroke are alsowithin scope of the invention, the methods comprising determining thepresence or absence of at least one allele of at least one polymorphicmarker in a nucleic acid sample obtained from the individual, whereinthe at least one polymorphic marker is selected from the markers setforth in Table 9, and markers in linkage disequilibrium therewith,wherein determination of the presence of the at least one allele isindicative of the treatment outcome of the individual.

In particular embodiments of the invention, e.g. in the various methods,uses, procedures, apparatus and kits of the invention, the cardiacarrhythmia phenotype is further characterized as being atrialfibrillation or atrial flutter. The inventors have determined that therisk conferred by the AF at-risk variants described herein is greaterfor individual with early age at onset than for individuals with lateage at onset. Thus in one embodiment, the atrial fibrillation or atrialflutter is further characterized by an age of onset in the individual ofless than 80 years. In another embodiment, the atrial fibrillation oratrial flutter is further characterized by an age of onset in theindividual of less than 70 years. In yet another embodiment, the atrialfibrillation or atrial flutter is further characterized by an age ofonset in the individual of less than 60 years. Other age cutoffs arepossible in alternative embodiments of the invention, and are alsocontemplated, including, but not limited to, age cutoff of less than 75years, less than 65 years, and less than 55 years. Furthermore, age atonset or diagnosis above age 55, 60, 65, 70, 75 or 80 are alsocontemplated and within scope of the invention, as are age ranges withinwhich diagnosis or symptoms or onset of the disease occurs, including,but not limited to, age 50-80, age 55-75, age 60-80, age 65-75, etc.

In certain embodiments of the invention, the stroke is furthercharacterized as ischemic stroke. In other embodiments, the strokephenotype may be characterized as one or more of the ischemic strokesub-phenotypes large artery atherosclerosis (LAA), cardioembolic stroke(CES) and small vessel disease (SVD).

In particular embodiments of the invention, linkage disequilibrium (LD)is defined by a specific quantitative cutoff. As described in detailherein, linkage disequilibrium can be quantitatively determined bymeasures such as r² and |D′|. As a consequence, certain embodiments ofthe invention relate to markers in linkage disequilibrium by a measurewithin a certain range specified by particular values of r² and/or |D′|.In one such embodiment, LD is characterized by numerical values for r²of greater than 0.1. In another embodiment, LD is characterized bynumerical values for r² of greater than 0.1. In another embodiment, LDis characterized by numerical values for r² of greater than 0.5. In yetanother embodiment, LD is characterized by numerical values for r² ofgreater than 0.8. Other cutoff values for r² are also contemplated, asdescribed in more detail herein. In certain embodiments, LD ischaracterized by certain cutoff values for r² and/or |D′|. In one suchembodiment, LD is characterized by values for r² and/or |D′| of greaterthan 0.2 and 0.8, respectively. Other combination and permutations ofthese or other measures of LD are possible to practice the invention,and are also contemplated and within scope of the invention.

The procedures, uses, or methods of the invention in some embodimentsfurther comprise a step of administering to an individual determined tobe at increased risk for developing cardiac arrhythmia or stroke acomposition comprising at least one therapeutic agent effective to treator prevent cardiac arrhythmia or stroke, or prevent symptoms associatedwith cardiac arrhythmia or stroke. Thus, the invention can be used todetermine whether an individual is suitable for a particular treatmentmodule.

Kits for use in the various methods and procedures described herein arealso within scope of the invention. Thus, in one aspect, the inventionrelates to a kit for assessing susceptibility to cardiac arrhythmiaand/or stroke in a human individual, the kit comprising reagents forselectively detecting at least one allele of at least one polymorphicmarker in the genome of the individual, wherein the polymorphic markeris selected from the group consisting of the polymorphic markers withinthe segment whose sequence is set forth in SEQ ID NO:50, and markers inlinkage disequilibrium therewith, and wherein the presence of the atleast one allele is indicative of a susceptibility to cardiac arrhythmiaand/or stroke.

In one embodiment, the at least one polymorphic marker is selected fromthe markers set forth in Table 5. In another embodiment, the at leastone polymorphic marker is selected from the group of markers set forthin Table 9, and markers in linkage disequilibrium therewith. In anotherembodiment, the at least one polymorphic marker is selected from markerrs2220427 (SEQ ID NO:1) and rs10033464 (SEQ ID NO:41), and markers inlinkage disequilibrium therewith. In one preferred embodiment, the atleast one polymorphic marker is selected from the markers set forth inTable 19. In another preferred embodiment, the at least one polymorphicmarker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33),rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ IDNO:41), and rs13143308 (SEQ ID NO:51). In one embodiment, the reagentscomprise at least one contiguous oligonucleotide that hybridizes to afragment of the genome of the individual comprising the at least onepolymorphic marker, a buffer and a detectable label.

In another embodiment, the reagents comprise at least one pair ofoligonucleotides that hybridize to opposite strands of a genomic nucleicacid segment obtained from the subject, wherein each oligonucleotideprimer pair is designed to selectively amplify a fragment of the genomeof the individual that includes one polymorphic marker, and wherein thefragment is at least 30 base pairs in size. The at least oneoligonucleotide is in preferred embodiments completely complementary tothe genome of the individual. In one embodiment, the oligonucleotide isabout 18 to about 50 nucleotides in length. In another embodiment, theoligonucleotide is 20-30 nucleotides in length. In one preferredembodiment, the kit comprises:

-   -   a. a detection oligonucleotide probe that is from 5-100        nucleotides in length;    -   b. an enhancer oligonucleotide probe that is from 5-100        nucleotides in length; and    -   c. an endonuclease enzyme;        wherein the detection oligonucleotide probe specifically        hybridizes to a first segment of the nucleic acid whose        nucleotide sequence is given by SEQ ID NO: 2 that comprises at        least one polymorphic site; wherein the detection        oligonucleotide probe comprises a detectable label at its 3′        terminus and a quenching moiety at its 5′ terminus; wherein the        enhancer oligonucleotide is from 5-100 nucleotides in length and        is complementary to a second segment of the nucleotide sequence        that is 5′ relative to the oligonucleotide probe, such that the        enhancer oligonucleotide is located 3′ relative to the detection        oligonucleotide probe when both oligonucleotides are hybridized        to the nucleic acid; wherein a single base gap exists between        the first segment and the second segment, such that when the        oligonucleotide probe and the enhancer oligonucleotide probe are        both hybridized to the nucleic acid, a single base gap exists        between the oligonucleotides; and wherein treating the nucleic        acid with the endonuclease will cleave the detectable label from        the 3′ terminus of the detection probe to release free        detectable label when the detection probe is hybridized to the        nucleic acid.

The polymorphic markers described herein as predictive of risk ofcardiac arrhythmia (e.g., AF and Atrial flutter) and stroke are usefulas diagnostic markers. In aspect, the invention therefore relates to theuse of an oligonucleotide probe in the manufacture of a diagnosticreagent for diagnosing and/or assessing susceptibility to cardiacarrhythmia and/or stroke in a human individual, wherein the probehybridizes to a segment of a nucleic acid whose nucleotide sequence isgiven by SEQ ID NO:50 that comprises at least one polymorphic site,wherein the fragment is 15-500 nucleotides in length.

In one such embodiment, the polymorphic site is selected from thepolymorphic markers set forth in Table 5, and polymorphisms in linkagedisequilibrium therewith. In another embodiment, the at least onepolymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073(SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1),rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51),

Computer-readable medium for storing information aboutdisease-associated markers as described herein are also within scope ofthe present invention. In one such aspect, the invention relates to acomputer-readable medium on which is stored an identifier for at leastone polymorphic marker; an indicator of the frequency of at least oneallele of said at least one polymorphic marker in a plurality ofindividuals diagnosed with atrial fibrillation, atrial flutter and/orstroke; and an indicator of the frequency of the least one allele ofsaid at least one polymorphic markers in a plurality of referenceindividuals; wherein the at least one polymorphic marker is selectedfrom the polymorphic markers set forth in Table 5, and polymorphisms inlinkage disequilibrium therewith. In a preferred embodiment, the atleast one polymorphic marker is selected from D4S406 (SEQ ID NO:45),rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ IDNO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51).

The invention also related to an apparatus for determining a geneticindicator for cardiac arrhythmia and/or stroke in a human individual,comprising: a computer readable memory; and a routine stored on thecomputer readable memory; wherein the routine is adapted to be executedon a processor to analyze genotype and/or haplotype data for at leastone human individual with respect to at least one polymorphic markerselected from the markers set forth in Table 5, and markers in linkagedisequilibrium therewith, and generate an output based on the marker orhaplotype data, wherein the output comprises a risk measure of the atleast one marker or haplotype as a genetic indicator of cardiacarrhythmia and/or stroke for the human individual. In a preferredembodiment, the routine further comprises determining an indicator ofthe frequency of at least one allele of at least one polymorphic markerand/or at least one haplotype in a plurality of individuals diagnosedwith cardiac arrhythmia and/or stroke, and an indicator of the frequencyof at the least one allele of at least one polymorphic marker or atleast one haplotype in a plurality of reference individuals, andcalculating a risk measure for the at least one allele and/or haplotypebased thereupon; and wherein a risk measure for the individual iscalculated based on a comparison of the at least one marker and/orhaplotype status for the individual to the calculated risk for the atleast one marker and/or haplotype information for the plurality ofindividuals diagnosed with atrial fibrillation, atrial flutter and/orstroke. In certain embodiments, the risk measure is characterized by anOdds Ratio (OR) or a Relative Risk (RR), as described in more detailherein.

The polymorphic markers discovered in the present invention aspredictive of a susceptibility of cardiac arrhythmia and stroke, asdescribed, as well as markers in linkage disequilibrium therewith, areall useful for practicing the various aspects of the present invention.Thus, although particular polymorphic markers were used by the presentinventors do detect an association of a particular region on chromosome4 to cardiac arrhythmia (e.g, atrial fibrillation and atrial flutter)and stroke, it is equally useful to assess markers in strong linkagedisequilibrium with those markers. As a consequence, in one embodimentof the methods, uses, kits, procedures, apparatus and media of theinvention, the at least one polymorphic marker or haplotype useful inthe methods or procedure of the invention comprises at least one of themarkers set forth in Table 5 (e.g., Table 5A and Table 5B) and markersin linkage disequilibrium therewith. In another embodiment, the at leastone polymorphic marker or haplotype comprises at least one of themarkers set forth in Table 9, and markers in linkage disequilibriumtherewith. In one embodiment, the at least one polymorphic marker orhaplotype comprises at least one of the markers set forth in Table 5. Inanother embodiment, the at least one polymorphic marker or haplotypecomprises at least one of the markers set forth in Table 9. In anotherembodiment, the at least one polymorphic marker is selected from themarkers set forth in Table 4. In one embodiment, the at least one markeris selected from marker rs2220427 (SEQ ID NO:1) and marker rs10033464(SEQ ID NO:41), and markers in linkage disequilibrium therewith. Inanother embodiment, the at least one polymorphic marker is selected fromthe markers set forth in Table 19.

In one embodiment, the at least one marker or haplotype comprises atleast one of markers D4S406 (SEQ ID NO:45), rs2723296 (SEQ ID NO:35),rs16997168 (SEQ ID NO:36), rs2723316 (SEQ ID NO:37), rs6419178 (SEQ IDNO:38), rs1448817 (SEQ ID NO:39), rs2634073 (SEQ ID NO:33), rs2200733(SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs13105878 (SEQ ID NO: 40),rs10033464 (SEQ ID NO:41), rs13141190 (SEQ ID NO:42), rs3853444 (SEQ IDNO:43), and rs4576077 (SEQ ID NO:44). In another embodiment, the atleast one marker or haplotype comprises at least one of the markersD4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ IDNO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), andrs13143308 (SEQ ID NO:51), In yet another embodiment, the at least onemarker is selected from rs10033464, rs2200733, rs13143308 and rs2220427,and markers in linkage disequilibrium therewith.

In a further embodiment, the presence of alleles-2, -4 and/or -8 ofmarker D4S406, allele G of marker rs2723296, allele T of markerrs16997168, allele T of marker rs2723316, allele A of marker rs6419178,allele G of marker rs1448817, allele A of marker rs2634073, allele T ofmarker rs2200733, allele T of marker rs2220427, allele C of markerrs13105878, allele T of marker rs10033464, allele A of markerrs13141190, allele A of marker rs3853444, and/or allele T of markerrs4576077 is indicative of increased susceptibility of cardiacarrhythmia or stroke in the individual.

In particular embodiments of the invention, the susceptibility conferredby the at-risk variant (i.e. a particular allele at a polymorphic marker(e.g, a SNP) or a particular haplotype) is increased susceptibility,i.e. the markers and haplotypes of the invention confer increased riskof an individual develops cardiac arrhtythmia, including atrialfibrillation and atrial flutter, and stroke. Susceptibility is typicallycharacterized by the measure Odds Ratio (OR) or, alternatively, by aRelative Risk (RR). In one embodiment, the increased susceptibility ischaracterized by an odds ratio (OR) of at least 1.3. In anotherembodiment, the increased susceptibility is characterized by an oddsratio (OR) of at least 1.4. In another embodiment, the increasedsusceptibility characterized by an odds ratio (OR) of at least 1.5. Inanother embodiment, the increased susceptibility characterized by anodds ratio (OR) or relative risk (RR) of at least 1.6. In yet anotherembodiment, the increased susceptibility characterized by an odds ratio(OR) or relative risk (RR) of at least 1.8. Other embodiments relate toother values for OR, or comparable values for RR including values of1.25, 1.35, 1.45, 1.55, etc.

Certain embodiments of the invention relate to Individuals of aparticular ethnicity or ancestry. In one such embodiment, the humanindividual has ancestry selected from black African ethnicity, Asianethnicity, Caucasian ethnicity, Hispanic ethnicity, and Arabicethnicity. In particular embodiments, the ethnicity is self-reported. Inother embodiments, ancestry is determined by the assessment ofparticular ethnicity-specific genetic markers.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention.

FIG. 1 Shows a plot of linkage disequilibrium (LD) in the regioncomprising variants of the present invention for the CEPH population(HapMap data). The LD block C04 (111,954,811-112,104,250 on Chromosome4, NCBI Build 35 positions) is indicated on the Figure by a black box.The plot shows two measures of LD, i.e. D′ in the upper and left part ofFIG. 1 and r² in the lower and right part of the figure.

FIG. 2 Shows a schematic of the haplotype structure at the associatedregion within the LD block. The areas of the dark (left) circles areproportional to the haplotype frequencies of the haplotypes in Icelandand the areas of the light (right) circles are proportional to thehaplotype frequencies in Hong Kong. The intermediary haplotype, shown inthe middle of the graph, no longer exists with certainty in either ofthe two populations (its estimated frequency is less than 0.2% which isindistinguishable from genotyping errors).

FIG. 3 Is an overview of a 200 kb genomic neighborhood of rs2200733 andrs10033464. It includes predicted ESTs, the locations of the three mainclasses of equivalent SNPs in the CEU HapMap samples and an overview ofthe LD structure of the region in the various ethnic HapMap samples.

FIG. 4. Shows Northern Blot analysis of PITX2 expression in human heartand aorta.

The PITX2 cDNA clone HU3_p983E0327D was used as a probe and detected1.8, 2 and 3 kb transcripts and 2.2 and 3 kb PITX2 transcripts in leftatrium and aorta respectively. Lane 1: Fetal heart, lane 2: Whole heart,lane 3: Aorta, lane 4: Apex of the heart, lane 5: Left atrium, lane 6:Right atrium, lane 7: Left ventricle lane 8: Right ventricle. Blotprobed with PITX2 cDNA clone (HU3_p983E0327D).

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

DEFINITIONS

The following terms shall, in the present context, have the meaning asindicated:

Atrial fibrillation (AF), as described herein, refers to AF as commonlydefined according to established medical criteria. AF classified byICD-10 in class 148 and by ICD-9 in class 427.3

Atrial flutter (AFI), as described herein, refers to AFI as commonlydefined according to established medical criteria. Afl is classifiedICD-10 class 148 and by ICD-9 in class 427.32.

A “polymorphic marker”, sometime referred to as a “marker”, as describedherein, refers to a genomic polymorphic site. Each polymorphic markerhas at least two sequence variations characteristic of particularalleles at the polymorphic site. Thus, genetic association to apolymorphic marker implies that there is association to at least onespecific allele of that particular polymorphic marker. The marker cancomprise any allele of any variant type found in the genome, includingSNPs, microsatellites, insertions, deletions, duplications andtranslocations.

An “allele” refers to the nucleotide sequence of a given locus(position) on a chromosome. A polymorphic marker allele thus refers tothe composition (i.e., sequence) of the marker on a chromosome. GenomicDNA from an individual contains two alleles for any given polymorphicmarker, representative of each copy of the marker on each chromosome.

A nucleotide position at which more than one sequence is possible in apopulation (either a natural population or a synthetic population, e.g.,a library of synthetic molecules) is referred to herein as a“polymorphic site”.

A “Single Nucleotide Polymorphism” or “SNP” is a DNA sequence variationoccurring when a single nucleotide at a specific location in the genomediffers between members of a species or between paired chromosomes in anindividual. Most SNP polymorphisms have two alleles. Each individual isin this instance either homozygous for one allele of the polymorphism(i.e. both chromosomal copies of the individual have the same nucleotideat the SNP location), or the individual is heterozygous (i.e. the twosister chromosomes of the individual contain different nucleotides). TheSNP nomenclature as reported herein refers to the official Reference SNP(rs) ID identification tag as assigned to each unique SNP by theNational Center for Biotechnological Information (NCBI).

A “variant”, as described herein, refers to a segment of DNA thatdiffers from the reference DNA. A “marker” or a “polymorphic marker”, asdefined herein, is a variant. Alleles that differ from the reference arereferred to as “variant” alleles.

A “microsatellite” is a polymorphic marker that has multiple smallrepeats of bases that are 2-8 nucleotides in length (such as CA repeats)at a particular site, in which the number of repeat lengths varies inthe general population. An “indel” is a common form of polymorphismcomprising a small insertion or deletion that is typically only a fewnucleotides long.

A “haplotype,” as described herein, refers to a segment of genomic DNAthat is characterized by a specific combination of alleles arrangedalong the segment. For diploid organisms such as humans, a haplotypecomprises one member of the pair of alleles for each polymorphic markeror locus. In a certain embodiment, the haplotype can comprise two ormore alleles, three or more alleles, four or more alleles, or five ormore alleles.

The term “susceptibility”, as described herein, encompasses bothincreased susceptibility and decreased susceptibility. Thus, particularpolymorphic markers and/or haplotypes of the invention may becharacteristic of increased susceptibility (i.e., increased risk) ofatrial fibrillation or stroke, as characterized by a relative risk (RR)or odds ratio (OR) of greater than one. Alternatively, the markersand/or haplotypes of the invention are characteristic of decreasedsusceptibility (i.e., decreased risk) of atrial fibrillation or stroke,as characterized by a relative risk of less than one.

A “nucleic acid sample” is a sample obtained from an individuals thatcontains nucleic acid. In certain embodiments, i.e. the detection ofspecific polymorphic markers and/or haplotypes, the nucleic acid samplecomprises genomic DNA. Such a nucleic acid sample can be obtained fromany source that contains genomic DNA, including as a blood sample,sample of amniotic fluid, sample of cerebrospinal fluid, or tissuesample from skin, muscle, buccal or conjunctival mucosa, placenta,gastrointestinal tract or other organs.

The term “atrial fibrillation and/or stroke therapeutic agent” refers toan agent that can be used to ameliorate or prevent symptoms associatedwith atrial fibrillation (AF), atrial flutter (AFI) or stroke, asdescribed in more detail herein.

The term “cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke-associated nucleic acid”, as described herein,refers to a nucleic acid that has been found to be associated to cardiacarrhythmia, e.g., atrial fibrillation (AF), atrial flutter (AFI) orstroke. This includes, but is not limited to, the markers and haplotypesdescribed herein and markers and haplotypes in strong linkagedisequilibrium (LD) therewith. In one embodiment, an atrialfibrillation, atrial flutter or stroke-associated nucleic acid refers tothe LD-block C04 found to be associated with atrial fibrillation andstroke. In another embodiment, the atrial fibrillation, atrial flutteror stroke-associated nucleic acid refers to the PITX2 gene.

The term “LD Block C04”, as described herein, refers to the LinkageDisequilibrium (LD) block on Chromosome 4 between position 111,954,811and 112,104,250 of NCBI (National Center for Biotechnology Information)Build 35, with the genomic sequence as set forth in SEQ ID NO:50.

The term “fragment”, as described herein, refers to a segment of anucleic acid or protein sequence. Fragments are of size smaller thantheir reference point, i.e. a fragment of a reference nucleic acidmolecule that is 1000 nucleotides in size is smaller than 1000nucleotides in size. Nucleic acid fragments of the invention arecommonly more than 5 nucleotides in size and typically more than 15nucleotides in size, with an upper limit as defined by either theirreference nucleotide or by the practical utility of the nucleotidefragment. For example, nucleotide fragments useful as hybridizationprobes in some embodiments of the invention are more than 15 nucleotidesand less than about 500 nucleotides in size. Other size ranges willapply for other nucleotide fragments and protein or peptide fragments ofthe invention.

The term “PITX2”, as described herein, refers to the paired-likehomeodomain transcription factor 2 gene on chromosome 4q25. This gene isalso referred to as pituitary homeobox 2 (PTX2), rieg bicoid-relatedhomeobox transcription factor 1 (RIEG1), solurshin, and all-1 responsivegene 1 (ARP1).

The present invention relates to the observation that certainpolymorphic markers on chromosome 4q25 of the human genome have beenfound to be associated with cardiac arrhythmia an stroke. In particularembodiments of the invention, polymorphic markers at chromosome 4q25 areassociated with the cardiac arrhythmias Atrial fibrillation (AF) andAtrial flutter (AFI), and stroke. These observation have important andunforeseen implications for the development of diagnostic andtherapeutics methods, uses, kits and systems, as described in furtherdetail herein.

In a genome-wide scan for genetic variants conferring susceptibility toAF, several markers on chromosome 4q25 were found to be associated withAF. The most significant association was found for markers rs2220427 andrs2220733, both of which gave p-values close to 10⁻⁹ (Table 2) for AF,and smaller, but nominally significant association to stroke (Table 3).A large number of markers were identified as perfect surrogates forthese markers, including the microsatellite marker D4S406 (Table 1) anda number of SNP markers (Table 4).

Further refinement of the results revealed that the association signalappears to center, in genetic terms, to markers of rs2200733 andrs10033464 (Table 7) and markers in linkage disequilibrium with thosemarkers (including, but not limited to, the SNP markers listed in Table9).

The original observation in the Icelandic population was replicated inan independent Icelandic AF/AFI cohort, in a Swedish AF cohort, and in aUS AF cohort (Table 7). When combined with the Icelandic samples, theassociation to rs2200733 was unequivocal (OR=1.72, P=3.3×10⁻⁴¹), and thesignificance of rs10033464 was well beyond the threshold of genome-widesignificance (OR=1.39, P=6.9×10⁻¹¹). Assuming the multiplicative model,the population attributable risk (PAR) of the two variants combined isapproximately 20% in populations of European ancestry. Furthermore, theassociation replicated in a Chinese AF cohort from Hong Kong (Table 7).

The inventors have also found that age at diagnosis of AF/AFI for theIcelandic samples correlates with the two SNPs rs2200733 and rs10033464.Thus, diagnosis occurs 2.28 years earlier per T allele of rs2200733 and1.10 years earlier per T allele of rs10033464 (joint P=1.29×10⁻⁶). Thiseffect is manifested by the association of the two variants beingstrongest in those diagnosed at a younger age, although the risk remainssignificant even in those diagnosed after reaching 80 years of age(Table 8). A similar age at onset effect is observed in the US cohort(Table 8).

The inventors have also observed a strong association between thevariants and AFI, that appears to be even stronger than for AF. Thus isrevealed by the association to the subset (N=116) of the Icelandicpatients that have a diagnosis of AFI (OR=2.60, 95% confidence interval(CI)=1.83-3.68, P=7.5×10⁻⁸ for rs2200733, OR=1.94, 95% CI=1.26-3.00,P=0.0028 for rs10033464). In fact, for rs2200733, the OR for thesedefinite AFI cases is significantly higher than that for the cases withan AF phenotype (P=0.0026), and close to significantly higher forrs10033464 (P=0.084). These results that both AF and AFI havesignificant genetic risk factors that are illustrated by the associationto SNPs rs2200733 and rs10033464.

The inventors have furthermore established that the variants associatingwith AF/AFI also associated with stroke, in particular ischemic stroke(Table 21). Marker rs2200733 replicated significantly in Ischemic strokeand in the Ischemic stroke (IS) subphenotype cardioembolic stroke (CES).Both this marker and marker rs10033464 were found, after genotypingadditional Icelandic IS cases and controls (total 1,943 cases/25,708controls) and four large IS case/control replication sets (4,294cases/3,709 controls), to associate most strongly with the CES, of whichAF is the primary cause, (rs2200733: OR=1.53, P=1.5×10⁻¹²; rs10033464:OR=1.27, P=5.9×10⁻⁴) (Table 21).

There is no known gene present in the LD block containing rs2200733 andrs10033464 (FIG. 3). The LD block contains one spliced EST (DA725631)and two single-exon ESTs (DB324364 and AF017091). RT-PCR of cDNAlibraries from various tissues did not detect the expression of theseESTs (Table 16). The PITX2 gene located in the adjacent upstream LDblock is the gene closest to the risk variants. Several markers withinthe LD block containing PITX2 gene are correlated to the markers showingassociation to AF and Afl, as shown in Table 18. It is thereforepossible that variants within the PITX2 gene are the underlyingcausative variants. Alternatively, it is possible that the variants ofthe present invention, as described herein, affect the function,stability, expression, post-translational modification, splicing,message stability of PITX2, or by other means affect the gene so as topredispose to the symptoms associated with atrial fibrillation, atrialflutter and/or stroke. The protein encoded by this gene, the paired-likehomeodomain transcription factor 2, is an interesting candidate forAF/AFI as it is known to play an important role in cardiac developmentby directing asymmetric morphogenesis of the heart (Franco, D., TrendsCardiovasc Med 13: 157-63 (2003)). Furthermore, in a mouse knockoutmodel Pitx2 has been shown to suppress a default pathway for sinoatrialnode formation in the left atrium. There is very little mRNA expressionof PITX2 in all easily accessible tissues, such as blood and adiposetissue, hampering the study of correlation between genotypes andexpression levels. The next gene upstream of PITX2 is ENPEP, anaminopeptidase responsible for the breakdown of angiotensin II in thevascular endothelium. This gene is expressed more widely, but thevariants associated with AF showed no correlation to its expression inblood or adipose tissue. No other annotated genes are located within a400 kb region upstream and 1.5 Mb regions downstream of the associatedvariants.

Assessment for Markers and Haplotypes

The genomic sequence within populations is not identical whenindividuals are compared. Rather, the genome exhibits sequencevariability between individuals at many locations in the genome. Suchvariations in sequence are commonly referred to as polymorphisms, andthere are many such sites within each genome For example, the humangenome exhibits sequence variations which occur on average every 500base pairs. The most common sequence variant consists of base variationsat a single base position in the genome, and such sequence variants, orpolymorphisms, are commonly called Single Nucleotide Polymorphisms(“SNPs”). These SNPs are believed to have occurred in a singlemutational event, and therefore there are usually two possible allelespossible at each SNPsite; the original allele and the mutated allele.Due to natural genetic drift and possibly also selective pressure, theoriginal mutation has resulted in a polymorphism characterized by aparticular frequency of its alleles in any given population. Many othertypes of sequence variants are found in the human genome, includingmicrosatellites, insertions, deletions, inversions and copy numbervariations. A polymorphic microsatellite has multiple small repeats ofbases (such as CA repeats, TG on the complimentary strand) at aparticular site in which the number of repeat lengths varies in thegeneral population. In general terms, each version of the sequence withrespect to the polymorphic site represents a specific allele of thepolymorphic site. These sequence variants can all be referred to aspolymorphisms, occurring at specific polymorphic sites characteristic ofthe sequence variant in question. In general terms, polymorphisms cancomprise any number of specific alleles. Thus in one embodiment of theinvention, the polymorphism is characterized by the presence of two ormore alleles in any given population. In another embodiment, thepolymorphism is characterized by the presence of three or more alleles.In other embodiments, the polymorphism is characterized by four or morealleles, five or more alleles, six or more alleles, seven or morealleles, nine or more alleles, or ten or more alleles. All suchpolymorphisms can be utilized in the methods and kits of the presentinvention, and are thus within the scope of the invention.

In some instances, reference is made to different alleles at apolymorphic site without choosing a reference allele. Alternatively, areference sequence can be referred to for a particular polymorphic site.The reference allele is sometimes referred to as the “wild-type” alleleand it usually is chosen as either the first sequenced allele or as theallele from a “non-affected” individual (e.g., an individual that doesnot display a trait or disease phenotype).

Alleles for SNP markers as referred to herein refer to the bases A, C, Gor T as they occur at the polymorphic site in the SNP assay employed.The allele codes for SNPs used herein are as follows: 1=A, 2=C, 3=G,4=T. The person skilled in the art will however realise that by assayingor reading the opposite DNA strand, the complementary allele can in eachcase be measured. Thus, for a polymorphic site (polymorphic marker)characterized by an A/G polymorphism, the assay employed may be designedto specifically detect the presence of one or both of the two basespossible, i.e. A and G. Alternatively, by designing an assay that isdesigned to detect the opposite strand on the DNA template, the presenceof the complementary bases T and C can be measured. Quantitatively (forexample, in terms of relative risk), identical results would be obtainedfrom measurement of either DNA strand (+ strand or − strand).

Typically, a reference sequence is referred to for a particularsequence. Alleles that differ from the reference are sometimes referredto as “variant” alleles. A variant sequence, as used herein, refers to asequence that differs from the reference sequence but is otherwisesubstantially similar. Alleles at the polymorphic genetic markersdescribed herein are variants. Additional variants can include changesthat affect a polypeptide. Sequence differences, when compared to areference nucleotide sequence, can include the insertion or deletion ofa single nucleotide, or of more than one nucleotide, resulting in aframe shift; the change of at least one nucleotide, resulting in achange in the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of a reading frame; duplication ofall or a part of a sequence; transposition; or a rearrangement of anucleotide sequence. Such sequence changes can alter the polypeptideencoded by the nucleic acid. For example, if the change in the nucleicacid sequence causes a frame shift, the frame shift can result in achange in the encoded amino acids, and/or can result in the generationof a premature stop codon, causing generation of a truncatedpolypeptide. Alternatively, a polymorphism associated with a disease ortrait can be a synonymous change in one or more nucleotides (i.e., achange that does not result in a change in the amino acid sequence).Such a polymorphism can, for example, alter splice sites, affect thestability or transport of mRNA, or otherwise affect the transcription ortranslation of an encoded polypeptide. It can also alter DNA to increasethe possibility that structural changes, such as amplifications ordeletions, occur at the somatic level. The polypeptide encoded by thereference nucleotide sequence is the “reference” polypeptide with aparticular reference amino acid sequence, and polypeptides encoded byvariant alleles are referred to as “variant” polypeptides with variantamino acid sequences. A sequence or a reference sequence can eitherrepresent the (+) or (−) direction of double stranded DNA. Suchsequences are related as being the reverse complement of one another, aswell known to the skilled person.

A haplotype refers to a segment of DNA that is characterized by aspecific combination of alleles arranged along the segment. For diploidorganisms such as humans, a haplotype comprises one member of the pairof alleles for each polymorphic marker or locus. In a certainembodiment, the haplotype can comprise two or more alleles, three ormore alleles, four or more alleles, or five or more alleles, each allelecorresponding to a specific polymorphic marker along the segment.Haplotypes can comprise a combination of various polymorphic markers,e.g., SNPs and microsatellites, having particular alleles at thepolymorphic sites. The haplotypes thus comprise a combination of allelesat various genetic markers.

Detecting specific polymorphic markers and/or haplotypes can beaccomplished by methods known in the art for detecting sequences atpolymorphic sites. For example, standard techniques for genotyping forthe presence of SNPs and/or microsatellite markers can be used, such asfluorescence-based techniques (Chen, X. et al., Genome Res. 9(5): 492-98(1999)), utilizing PCR, LCR, Nested PCR and other techniques for nucleicacid amplification. Specific methodologies available for SNP genotypinginclude, but are not limited to, TaqMan genotyping assays and SNPIexplatforms (Applied Biosystems), mass spectrometry (e.g., MassARRAYsystem from Sequenom), minisequencing methods, real-time PCR, Bio-Plexsystem (BioRad), CEQ and SNPstream systems (Beckman), MolecularInversion Probe array technology (e.g., Affymetrix GeneChip), andBeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays).By these or other methods available to the person skilled in the art,one or more alleles at polymorphic markers, including microsatellites,SNPs or other types of polymorphic markers, can be identified.

In certain methods described herein, an individual who is at anIncreased susceptibility (i.e., increased risk) for any specific diseaseor trait under study, is an individual in whom at least one specificallele at one or more polymorphic marker or haplotype conferringincreased susceptibility for the disease or trait is identified (i.e.,at-risk marker alleles or haplotypes). In one aspect, the at-risk markeror haplotype is one that confers a significant increased risk (orsusceptibility) of the disease or trait. In one embodiment, significanceassociated with a marker or haplotype is measured by a relative risk(RR). In another embodiment, significance associated with a marker orhaplotye is measured by an odds ratio (OR). In a further embodiment, thesignificance is measured by a percentage. In one embodiment, asignificant increased risk is measured as a risk (relative risk and/orodds ratio) of at least 1.2, including but not limited to: at least 1.2,at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7,1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least4.0, and at least 5.0. In a particular embodiment, a risk (relative riskand/or odds ratio) of at least 1.2 is significant. In another particularembodiment, a risk of at least 1.3 is significant. In yet anotherembodiment, a risk of at least 1.4 is significant. In a furtherembodiment, a relative risk of at least about 1.5 is significant. Inanother further embodiment, a significant increase in risk is at leastabout 1.7 is significant. However, other cutoffs are also contemplated,e.g. at least 1.15, 1.25, 1.35, and so on, and such cutoffs are alsowithin scope of the present invention. In other embodiments, asignificant increase in risk is at least about 20%, including but notlimited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, and 500%. In one particularembodiment, a significant increase in risk is at least 20%. In otherembodiments, a significant increase in risk is at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% and at least 100%. Other cutoffs or ranges as deemed suitable by theperson skilled in the art to characterize the invention are however alsocontemplated, and those are also within scope of the present invention.

An at-risk polymorphic marker or haplotype of the present invention isone where at least one allele of at least one marker or haplotype ismore frequently present in an individual at risk for the disease ortrait (e.g., cardiac arrhythmia or stroke) (affected), compared to thefrequency of its presence in a comparison group (control), and whereinthe presence of the marker or haplotype is indicative of susceptibilityto the disease or trait. The control group may in one embodiment be apopulation sample, i.e. a random sample from the general population. Inanother embodiment, the control group is represented by a group ofindividuals who are disease-free. Such disease-free control may in oneembodiment be characterized by the absence of one or more specificdisease-associated symptoms. In another embodiment, the disease-freecontrol group is characterized by the absence of one or moredisease-specific risk factors. Such risk factors are in one embodimentat least one environmental risk factor. Representative environmentalfactors are natural products, minerals or other chemicals which areknown to affect, or contemplated to affect, the risk of developing thespecific disease or trait. Other environmental risk factors are riskfactors related to lifestyle, including but not limited to food anddrink habits, geographical location of main habitat, and occupationalrisk factors. In another embodiment, the risk factors are at least onegenetic risk factor.

As an example of a simple test for correlation would be a Fisher-exacttest on a two by two table. Given a cohort of chromosomes, the two bytwo table is constructed out of the number of chromosomes that includeboth of the markers or haplotypes, one of the markers or haplotypes butnot the other and neither of the markers or haplotypes.

In other embodiments of the invention, an individual who is at adecreased susceptibility (i.e., at a decreased risk) for the disease ortrait is an individual in whom at least one specific allele at one ormore polymorphic marker or haplotype conferring decreased susceptibilityfor the disease or trait is identified. The marker alleles and/orhaplotypes conferring decreased risk are also said to be protective. Inone aspect, the protective marker or haplotype is one that confers asignificant decreased risk (or susceptibility) of the disease or trait.In one embodiment, significant decreased risk is measured as a relativerisk of less than 0.9, including but not limited to less than 0.9, lessthan 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4,less than 0.3, less than 0.2 and less than 0.1. In one particularembodiment, significant decreased risk is less than 0.7. In anotherembodiment, significant decreased risk is less than 0.5. In yet anotherembodiment, significant decreased risk is less than 0.3. In anotherembodiment, the decrease in risk (or susceptibility) is at least 20%,including but not limited to at least 25%, at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% and at least 98%. In one particular embodiment,a significant decrease in risk is at least about 30%. In anotherembodiment, a significant decrease in risk at least about 50%. Inanother embodiment, the decrease in risk is at least about 70%. Othercutoffs or ranges as deemed suitable by the person skilled in the art tocharacterize the invention are however also contemplated, and those arealso within scope of the present invention.

The person skilled in the art will appreciate that for markers with twoalleles present in the population being studied, and wherein one alleleis found in increased frequency in a group of individuals with a traitor disease in the population, compared with controls, the other alleleof the marker will be found in decreased frequency in the group ofindividuals with the trait or disease, compared with controls. In such acase, one allele of the marker (the one found in increased frequency inindividuals with the trait or disease) will be the at-risk allele, whilethe other allele will be a protective allele.

Linkage Disequilibrium

The natural phenomenon of recombination, which occurs on average oncefor each chromosomal pair during each meiotic event, represents one wayin which nature provides variations in sequence (and biological functionby consequence). It has been discovered that recombination does notoccur randombly in the genome; rather, there are large variations in thefrequency of recombination rates, resulting in small regions of highrecombination frequency (also called recombination hotspots) and largerregions of low recombination frequency, which are commonly referred toas Linkage Disequilibrium (LD) blocks (Myers, S. et al., Biochem SocTrans 34:526-530 (2006); Jeffreys, A. J., et al., Nature Genet29:217-222 (2001); May, C. A., et al., Nature Genet 31:272-275 (2002)).

Linkage Disequilibrium (LD) refers to a non-random assortment of twogenetic elements. For example, if a particular genetic element (e.g., anallele of a polymorphic marker, or a haplotype) occurs in a populationat a frequency of 0.25 (25%) and another element occurs at a frequencyof 0.25 (25%), then the predicted occurrence of a person's having bothelements is 0.125 (12.5%), assuming a random distribution of theelements. However, if it is discovered that the two elements occurtogether at a frequency higher than 0.125, then the elements are said tobe in linkage disequilibrium, since they tend to be inherited togetherat a higher rate than what their independent frequencies of occurrence(e.g., allele or haplotype frequencies) would predict. Roughly speaking,LD is generally correlated with the frequency of recombination eventsbetween the two elements. Allele or haplotype frequencies can bedetermined in a population by genotyping individuals in a population anddetermining the frequency of the occurrence of each allele or haplotypein the population. For populations of diploids, e.g., human populations,individuals will typically have two alleles for each genetic element(e.g., a marker, haplotype or gene).

Many different measures have been proposed for assessing the strength oflinkage disequilibrium (LD). Most capture the strength of associationbetween pairs of biallelic sites. Two important pairwise measures of LDare r² (sometimes denoted Δ²) and |D′|. Both measures range from 0 (nodisequilibrium) to 1 (‘complete’ disequilibrium), but theirinterpretation is slightly different. |D′| is defined in such a way thatit is equal to 1 if just two or three of the possible haplotypes arepresent, and it is <1 if all four possible haplotypes are present.Therefore, a value of |D′| that is <1 indicates that historicalrecombination may have occurred between two sites (recurrent mutationcan also cause |D′| to be <1, but for single nucleotide polymorphisms(SNPs) this is usually regarded as being less likely thanrecombination). The measure r² represents the statistical correlationbetween two sites, and takes the value of 1 if only two haplotypes arepresent.

The r² measure is arguably the most relevant measure for associationmapping, because there is a simple inverse relationship between r² andthe sample size required to detect association between susceptibilityloci and SNPs. These measures are defined for pairs of sites, but forsome applications a determination of how strong LD is across an entireregion that contains many polymorphic sites might be desirable (e.g.,testing whether the strength of LD differs significantly among loci oracross populations, or whether there is more or less LD in a region thanpredicted under a particular model). Measuring LD across a region is notstraightforward, but one approach is to use the measure r, which wasdeveloped in population genetics. Roughly speaking, r measures how muchrecombination would be required under a particular population model togenerate the LD that is seen in the data. This type of method canpotentially also provide a statistically rigorous approach to theproblem of determining whether LD data provide evidence for the presenceof recombination hotspots. For the methods and procedures describedherein, a significant r² value can be at least 0.1 such as at least 0.1,0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or1.0. In one preferred embodiment, the significant r² value can be atleast 0.2. Alternatively, linkage disequilibrium as described herein,refers to linkage disequilibrium characterized by values of |D′| of atleast 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96,0.97, 0.98, 0.99. Thus, linkage disequilibrium represents a correlationbetween alleles of distinct markers. It is measured by correlationcoefficient or |D′| (r² up to 1.0 and |D′| up to 1.0). Linkagedisequilibrium can be determined in a single human population, asdefined herein, or it can be determined in a collection of samplescomprising individuals from more than one human population. In oneembodiment of the invention, LD is determined in a sample from one ormore of the HapMap populations (caucasian, african, japanese, chinese),as defined (http://www.hapmap.org). In one such embodiment, LD isdetermined in the CEU population of the HapMap samples. In anotherembodiment, LD is determined in the YRI population. In yet anotherembodiment, LD is determined in samples from the Icelandic population.

If all polymorphisms in the genome were identical at the populationlevel, then every single one of them would need to be investigated inassociation studies. However, due to linkage disequilibrium betweenpolymorphisms, tightly linked polymorphisms are strongly correlated,which reduces the number of polymorphisms that need to be investigatedin an association study to observe a significant association. Anotherconsequence of LD is that many polymorphisms may give an associationsignal due to the fact that these polymorphisms are strongly correlated.

Genomic LD maps have been generated across the genome, and such LD mapshave been proposed to serve as framework for mapping disease-genes(Risch, N. & Merkiangas, K, Science 273:1516-1517 (1996); Maniatis, N.,et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, D E et al,Nature 411:199-204 (2001)).

It is now established that many portions of the human genome can bebroken into series of discrete haplotype blocks containing a few commonhaplotypes; for these blocks, linkage disequilibrium data provideslittle evidence indicating recombination (see, e.g., Wall., J. D. andPritchard, J. K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. etal., Nature Genet. 29:229-232 (2001); Gabriel, S. B. et al., Science296:2225-2229 (2002); Patil, N. et al., Science 294:1719-1723 (2001);Dawson, E. et al., Nature 418:544-548 (2002); Phillips, M. S. et al.,Nature Genet. 33:382-387 (2003)).

There are two main methods for defining these haplotype blocks: Blockscan be defined as regions of DNA that have limited haplotype diversity(see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N.et al., Science 294:1719-1723 (2001); Dawson, E. et al., Nature418:544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA99:7335-7339 (2002)), or as regions between transition zones havingextensive historical recombination, identified using linkagedisequilibrium (see, e.g., Gabriel, S. B. et al., Science 296:2225-2229(2002); Phillips, M. S. et al., Nature Genet. 33:382-387 (2003); Wang,N. et al., Am. J. Hum. Genet. 71:1227-1234 (2002); Stumpf, M. P., andGoldstein, D. B., Curr. Biol. 13:1-8 (2003)). More recently, afine-scale map of recombination rates and corresponding hotspots acrossthe human genome has been generated (Myers, S., et al., Science310:321-32324 (2005); Myers, S. et al., Biochem Soc Trans 34:526530(2006)). The map reveals the enormous variation in recombination acrossthe genome, with recombination rates as high as 10-60 cM/Mb in hotspots,while closer to 0 in intervening regions, which thus represent regionsof limited haplotype diversity and high LD. The map can therefore beused to define haplotype blocks/LD blocks as regions flanked byrecombination hotspots. As used herein, the terms “haplotype block” or“LD block” includes blocks defined by any of the above describedcharacteristics, or other alternative methods used by the person skilledin the art to define such regions.

Haplotype blocks can be used to map associations between phenotype andhaplotype status, using single markers or haplotypes comprising aplurality of markers. The main haplotypes can be identified in eachhaplotype block, and then a set of “tagging” SNPs or markers (thesmallest set of SNPs or markers needed to distinguish among thehaplotypes) can then be identified. These tagging SNPs or markers canthen be used in assessment of samples from groups of individuals, inorder to identify association between phenotype and haplotype. Ifdesired, neighboring haplotype blocks can be assessed concurrently, asthere may also exist linkage disequilibrium among the haplotype blocks.

It has thus become apparent that for any given observed association to apolymorphic marker in the genome, it is likely that additional markersin the genome also show association. This is a natural consequence ofthe uneven distribution of LD across the genome, as observed by thelarge variation in recombination rates. The markers used to detectassociation thus in a sense represent “tags” for a genomic region (i.e.,a haplotype block or LD block) that is associating with a given diseaseor trait, and as such are useful for use in the methods and kits of thepresent invention. One or more causative (functional) variants ormutations may reside within the region found to be associating to thedisease or trait. Such variants may confer a higher relative risk (RR)or odds ratio (OR) than observed for the tagging markers used to detectthe association. The present invention thus refers to the markers usedfor detecting association to the disease, as described herein, as wellas markers in linkage disequilibrium with the markers. Thus, in certainembodiments of the invention, markers that are in LD with the markersand/or haplotypes of the invention, as described herein, may be used assurrogate markers. The surrogate markers have in one embodiment relativerisk (RR) and/or odds ratio (OR) values smaller than for the markers orhaplotypes initially found to be associating with the disease, asdescribed herein. In other embodiments, the surrogate markers have RR orOR values greater than those initially determined for the markersinitially found to be associating with the disease, as described herein.An example of such an embodiment would be a rare, or relatively rare(<10% allelic population frequency) variant in LD with a more commonvariant (>10% population frequency) initially found to be associatingwith the disease, such as the variants described herein. Identifying andusing such markers for detecting the association discovered by theinventors as described herein can be performed by routine methods wellknown to the person skilled in the art, and are therefore within thescope of the present invention.

It is possible that certain polymorphic markers in linkagedisequilibrium with the markers shown herein to be associated withcardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) andstroke are located outside the physical boundaries of the LD block C04as defined herein by the sequence set forth in SEQ ID NO:50. This is aconsequence of the historical recombination rates in the region inquestion, which may have led to a region of strong LD (the LD block),with residual markers outside the block in LD with markers within theblock. Such markers are also within scope of the present invention, asthey are also useful for practicing the invention by virtue of theirgenetic relationship with the markers shown herein to be associated withcardiac arrhythmia and stroke. Examples of such markers are shown inTable 18 (rs7668322 (SEQ ID NO:46), rs2197815 (SEQ ID NO:47), rs6831623(SEQ ID NO:48), rs2595110 (SEQ ID NO:49))

Determination of Haplotype Frequency

The frequencies of haplotypes in patient and control groups can beestimated using an expectation-maximization algorithm (Dempster A. etal., J. R. Stat. Soc. B, 39: 1-38 (1977)). An implementation of thisalgorithm that can handle missing genotypes and uncertainty with thephase can be used. Under the null hypothesis, the patients and thecontrols are assumed to have identical frequencies. Using a likelihoodapproach, an alternative hypothesis is tested, where a candidateat-risk-haplotype, which can include the markers described herein, isallowed to have a higher frequency in patients than controls, while theratios of the frequencies of other haplotypes are assumed to be the samein both groups. Likelihoods are maximized separately under bothhypotheses and a corresponding 1-df likelihood ratio statistic is usedto evaluate the statistical significance.

To look for at-risk and protective markers and haplotypes within alinkage region, for example, association of all possible combinations ofgenotyped markers is studied, provided those markers span a practicalregion. The combined patient and control groups can be randomly dividedinto two sets, equal in size to the original group of patients andcontrols. The marker and haplotype analysis is then repeated and themost significant p-value registered is determined. This randomizationscheme can be repeated, for example, over 100 times to construct anempirical distribution of p-values. In a preferred embodiment, a p-valueof <0.05 is indicative of an significant marker and/or haplotypeassociation.

Haplotype Analysis

One general approach to haplotype analysis involves usinglikelihood-based inference applied to NEsted MOdels (Gretarsdottir S.,et al., Nat. Genet. 35: 131-38 (2003)). The method is implemented in theprogram NEMO, which allows for many polymorphic markers, SNPs andmicrosatellites. The method and software are specifically designed forcase-control studies where the purpose is to identify haplotype groupsthat confer different risks. It is also a tool for studying LDstructures. In NEMO, maximum likelihood estimates, likelihood ratios andp-values are calculated directly, with the aid of the EM algorithm, forthe observed data treating it as a missing-data problem.

Even though likelihood ratio tests based on likelihoods computeddirectly for the observed data, which have captured the information lossdue to uncertainty in phase and missing genotypes, can be relied on togive valid p-values, it would still be of interest to know how muchinformation had been lost due to the information being incomplete. Theinformation measure for haplotype analysis is described in Nicolae andKong (Technical Report 537, Department of Statistics, University ofStatistics, University of Chicago; Biometrics, 60(2):368-75 (2004)) as anatural extension of information measures defined for linkage analysis,and is implemented in NEMO.

For single marker association to a disease, the Fisher exact test can beused to calculate two-sided p-values for each individual allele.Usually, all p-values are presented unadjusted for multiple comparisonsunless specifically indicated. The presented frequencies (formicrosatellites, SNPs and haplotypes) are allelic frequencies as opposedto carrier frequencies. To minimize any bias due the relatedness of thepatients who were recruited as families for the linkage analysis, firstand second-degree relatives can be eliminated from the patient list.Furthermore, the test can be repeated for association correcting for anyremaining relatedness among the patients, by extending a varianceadjustment procedure described in Risch, N. & Teng, J. (Genome Res.,8:1273-1288 (1998)), DNA pooling (ibid) for sibships so that it can beapplied to general familial relationships, and present both adjusted andunadjusted p-values for comparison. The differences are in general verysmall as expected. To assess the significance of single-markerassociation corrected for multiple testing we can carry out arandomization test using the same genotype data. Cohorts of patients andcontrols can be randomized and the association analysis redone multipletimes (e.g., up to 500,000 times) and the p-value is the fraction ofreplications that produced a p-value for some marker allele that islower than or equal to the p-value we observed using the originalpatient and control cohorts.

For both single-marker and haplotype analyses, relative risk (RR) andthe population attributable risk (PAR) can be calculated assuming amultiplicative model (haplotype relative risk model) (Terwilliger, J. D.& Ott, J., Hum. Hered. 42:337-46 (1992) and Falk, C. T. & Rubinstein, P,Ann. Hum. Genet. 51 (Pt 3):227-33 (1987)), i.e., that the risks of thetwo alleles/haplotypes a person carries multiply. For example, if RR isthe risk of A relative to a, then the risk of a person homozygote AAwill be RR times that of a heterozygote Aa and RR² times that of ahomozygote aa. The multiplicative model has a nice property thatsimplifies analysis and computations—haplotypes are independent, i.e.,in Hardy-Weinberg equilibrium, within the affected population as well aswithin the control population. As a consequence, haplotype counts of theaffecteds and controls each have multinomial distributions, but withdifferent haplotype frequencies under the alternative hypothesis.Specifically, for two haplotypes, h_(i) and h_(j),risk(h_(i))/risk(h_(j))=(f_(i)/p_(i))/(f_(j)/p_(j)), where f and pdenote, respectively, frequencies in the affected population and in thecontrol population. While there is some power loss if the true model isnot multiplicative, the loss tends to be mild except for extreme cases.Most importantly, p-values are always valid since they are computed withrespect to null hypothesis.

Linkage Disequilibrium Using NEMO

LD between pairs of markers can be calculated using the standarddefinition of D′ and r² (Lewontin, R., Genetics 49:49-67 (1964); Hill,W. G. & Robertson, A. Theor. Appl. Genet. 22:226-231 (1968)). UsingNEMO, frequencies of the two marker allele combinations are estimated bymaximum likelihood and deviation from linkage equilibrium is evaluatedby a likelihood ratio test. The definitions of D′ and r² are extended toinclude microsatellites by averaging over the values for all possibleallele combination of the two markers weighted by the marginal alleleprobabilities. When plotting all marker combination to elucidate the LDstructure in a particular region, we plot D′ in the upper left cornerand the p-value in the lower right corner. In the LD plots the markerscan be plotted equidistant rather than according to their physicallocation, if desired.

Risk Assessment and Diagnostics

As described herein, certain polymorphic markers and haplotypescomprising such markers are found to be useful for risk assessment ofcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) orstroke. Risk assessment can involve the use of the markers fordiagnosing a susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) or stroke. Particular alleles ofpolymorphic markers are found more frequently in individuals withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) orstroke, than in individuals without diagnosis of cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) or stroke. Therefore,these marker alleles have predictive value for detecting cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) or stroke, in an individual. Tagging markers withinhaplotype blocks or LD blocks comprising at-risk markers, such as themarkers of the present invention, can be used as surrogates for othermarkers and/or haplotypes within the haplotype block or LD block.Markers with values of r² equal to 1 are perfect surrogates for theat-risk variants, i.e. genotypes for one marker perfectly predictsgenotypes for the other. Markers with smaller values of r² than 1 canalso be surrogates for the at-risk variant, or alternatively representvariants with relative risk values as high or possibly even higher thanthe at-risk variant. The at-risk variant identified may not be thefunctional variant itself, but is in this instance in linkagedisequilibrium with the true functional variant. The present inventionencompasses the assessment of such surrogate markers for the markers asdisclosed herein. Such markers are annotated, mapped and listed inpublic databases, as well known to the skilled person, or canalternatively be readily identified by sequencing the region or a partof the region identified by the markers of the present invention in agroup of individuals, and identify polymorphisms in the resulting groupof sequences. As a consequence, the person skilled in the art canreadily and without undue experimentation genotype surrogate markers inlinkage disequilibrium with the markers and/or haplotypes as describedherein. The tagging or surrogate markers in LD with the at-risk variantsdetected, also have predictive value for detecting association tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke or a susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) or stroke, in an individual. Thesetagging or surrogate markers that are in LD with the markers of thepresent invention can also include other markers that distinguish amonghaplotypes, as these similarly have predictive value for detectingsusceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke.

The markers and haplotypes of the invention, e.g., the markers presentedin Tables 5 and 9, as well as markers in linkage disequilibriumtherewith, may be useful for risk assessment and diagnostic purposesfor, either alone or in combination. Thus, even in cases where theincrease in risk by individual markers is relatively modest, i.e. on theorder of 10-30%, the association may have significant implications.Thus, relatively common variants may have significant contribution tothe overall risk (Population Attributable Risk is high), or combinationof markers can be used to define groups of individual who, based on thecombined risk of the markers, is at significant combined risk ofdeveloping the disease.

Thus, in one embodiment of the invention, a plurality of variants(markers and/or haplotypes) is used for overall risk assessment. Thesevariants are in one embodiment selected from the variants as disclosedherein. Other embodiments include the use of the variants of the presentinvention in combination with other variants known to be useful fordiagnosing a susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. In such embodiments, thegenotype status of a plurality of markers and/or haplotypes isdetermined in an individual, and the status of the individual comparedwith the population frequency of the associated variants, or thefrequency of the variants in clinically healthy subjects, such asage-matched and sex-matched subjects. Methods known in the art, such asmultivariate analyses or joint risk analyses, may subsequently be usedto determine the overall risk conferred based on the genotype status atthe multiple loci. Assessment of risk based on such analysis maysubsequently be used in the methods and kits of the invention, asdescribed herein.

As described in the above, the haplotype block structure of the humangenome has the effect that a large number of variants (markers and/orhaplotypes) in linkage disequilibrium with the variant originallyassociated with a disease or trait may be used as surrogate markers forassessing association to the disease or trait. The number of suchsurrogate markers will depend on factors such as the historicalrecombination rate in the region, the mutational frequency in the region(i.e., the number of polymorphic sites or markers in the region), andthe extent of LD (size of the LD block) in the region. These markers areusually located within the physical boundaries of the LD block orhaplotype block in question as defined using the methods describedherein, or by other methods known to the person skilled in the art.However, sometimes marker and haplotype association is found to extendbeyond the physical boundaries of the haplotype block as defined. Suchmarkers and/or haplotypes may in those cases be also used as surrogatemarkers and/or haplotypes for the markers and/or haplotypes physicallyresiding within the haplotype block as defined. As a consequence,markers and haplotypes in LD (typically characterized by r² greater than0.1, such as r² greater than 0.2, including r² greater than 0.3, alsoincluding r² greater than 0.4) with the markers and haplotypes of thepresent invention are also within the scope of the invention, even ifthey are physically located beyond the boundaries of the haplotype blockas defined. This includes markers that are described herein (e.g.,Tables 5 and 9), but may also include other markers that are in strongLD (characterized by r² greater than 0.1 or 0.2 and/or |D′|>0.8) withone or more of the markers listed in Tables 5 and 9.

For the SNP markers described herein, the opposite allele to the allelefound to be in excess in patients (at-risk allele) is found in decreasedfrequency in cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke patients. These markers and haplotypes in LDand/or comprising such markers, are thus protective for cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.i.e. they confer a decreased risk or susceptibility of individualscarrying these markers and/or haplotypes developing cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke.

Certain variants of the present invention, including certain haplotypescomprise, in some cases, a combination of various genetic markers, e.g.,SNPs and microsatellites. Detecting haplotypes can be accomplished bymethods known in the art and/or described herein for detecting sequencesat polymorphic sites. Furthermore, correlation between certainhaplotypes or sets of markers and disease phenotype can be verifiedusing standard techniques. A representative example of a simple test forcorrelation would be a Fisher-exact test on a two by two table.

In specific embodiments, a marker or haplotype found to be associatedwith cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke, (e.g., markers as listed in Table 5 (Tables 5A and 5B),Table 9 and/or Table 19, and markers in linkage disequilibriumtherewith) is one in which the marker allele or haplotype is morefrequently present in an individual at risk for cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke (affected),compared to the frequency of its presence in a healthy individual(control), wherein the presence of the marker allele or haplotype isindicative of cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke. or a susceptibility to cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke. In otherembodiments, at-risk markers in linkage disequilibrium with one or moremarkers found to be associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. (e.g., marker alleles aslisted in Tables 5A and 5B, and markers in linkage disequilibriumtherewith) are tagging markers that are more frequently present in anindividual at risk for cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke (affected), compared to the frequency oftheir presence in a healthy individual (control), wherein the presenceof the tagging markers is indicative of increased susceptibility tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke. In a further embodiment, at-risk markers alleles (i.e.conferring increased susceptibility) in linkage disequilibrium with oneor more markers found to be associated with cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke. (e.g., markeralleles as listed in Tables 5A and 5B and markers in linkagedisequilibrium therewith), are markers comprising one or more allelethat is more frequently present in an individual at risk for cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokecompared to the frequency of their presence in a healthy individual(control), wherein the presence of the markers is Indicative ofincreased susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke.

Study Population

In a general sense, the methods and kits of the invention can beutilized from samples containing genomic DNA from any source, i.e. anyindividual. In preferred embodiments, the individual is a humanindividual. The individual can be an adult, child, or fetus. The presentinvention also provides for assessing markers and/or haplotypes inindividuals who are members of a target population. Such a targetpopulation is in one embodiment a population or group of individuals atrisk of developing the disease, based on other genetic factors,biomarkers, biophysical parameters (e.g., weight, BMD, blood pressure),or general health and/or lifestyle parameters (e.g., history of diseaseor related diseases, previous diagnosis of disease, family history ofdisease).

The invention provides for embodiments that include individuals fromspecific age subgroups, such as those over the age of 40, over age of45, or over age of 50, 55, 60, 65, 70, 75, 80, or 85. Other embodimentsof the invention pertain to other age groups, such as individuals agedless than 85, such as less than age 80, less than age 75, or less thanage 70, 65, 60, 55, 50, 45, 40, 35, or age 30. Other embodiments relateto individuals with age at onset of the disease in any of the age rangesdescribed in the above. It is also contemplated that a range of ages maybe relevant in certain embodiments, such as age at onset at more thanage 45 but less than age 60. Other age ranges are however alsocontemplated, including all age ranges bracketed by the age valueslisted in the above.

Other embodiments related to individuals with age at onset of thedisease at particular age or age range. Thus, it is known thatpredisposing factors, genetic and non-genetic, can affect at what age anindividual develops a disease. For cardiovascular disorders, includingcardiac arrhythmias and stroke, common risk factors can influence if,and at what age, an individual develops the disease. Some embodiments ofthe invention therefore relate to age at onset or age at diagnosis ofcardiac arrhythmia (e.g., atrial fibrillation and/or atrial flutter) orstroke in a certain age range. In one embodiment, the individuals atrisk for developing cardiac arrhythmia (e.g., atrial fibrillation and/oratrial flutter) or stroke have age at onset or age at diagnosis over theage of 40. In other embodiments, the individuals have age at onset orage at diagnosis over age of 45, or over age of 50, 55, 60, 65, 70, 75,80, or 85. Other embodiments of the invention pertain to individuals whohave an age at onset or age at diagnosis at age less than 85, such asless than age 80, less than age 75, or less than age 70, 65, 60, 55, 50,45, 40, 35, or age 30. One preferred embodiment includes individualsdiagnosed with atrial fibrillation or atrial flutter or stroke below age80. Another preferred embodiment relates to individuals diagnosed withatrial fibrillation or atrial flutter or stroke below age 70. Anotherpreferred embodiment, relates to individuals diagnosed with atrialfibrillation or atrial flutter or stroke below age 60. Yet anotherpreferred embodiment relates to individuals diagnosed with atrialfibrillation or atrial flutter or stroke below age 50. Other embodimentsrelate to individuals with age at onset of the disease in specific ageranges, described in the above. It is also contemplated that a range ofages may be relevant in certain embodiments, such as age at onset atmore than age 45 but less than age 60, age at onset at age more than 60and less than age 70, age at onset at age more than 70 and less than 80,or age at onset at age more than 60 and less than 80. Other age rangesare however also contemplated, including all age ranges bracketed by theage values listed in the above.

The invention furthermore relates to individuals of either sex, males orfemales. It also provides for embodiments that relate to human subjectsthat are from one or more human population including, but not limitedto, Bantu, Mandenk, Yoruba, San, Mbuti Pygmy, Orcadian, Adygel, Russian,Sardinian, Tuscan, Mozabite, Bedouin, Druze, Palestinian, Balochi,Brahui, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash, Han,Dai, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi,Mongolan, Naxi, Cambodian, Japanese, Yakut, Melanesian, Papuan,Karitianan, Surui, Colmbian, Maya and Pima. The invention also relatesto European populations, American populations, Eurasian populations,Asian populations, Central/South Asian populations, East Asianpopulations, Middle Eastern populations, African populations, Hispanicpopulations, and Oceanian populations. European populations include, butare not limited to, Swedish, Norwegian, Finnish, Russian, Danish,Icelandic, Irish, Kelt, English, Scottish, Dutch, Belgian, French,German, Spanish, Portuguese, Italian, Polish, Bulgarian, Slavic,Serbian, Bosnian, Chech, Greek and Turkish populations.

In one preferred embodiment, the invention relates to populations thatinclude black African ancestry such as populations comprising persons ofAfrican descent or lineage. Black African ancestry may be determined byself reporting as African-Americans, Afro-Americans, Black Americans,being a member of the black race or being a member of the negro race.For example, African Americans or Black Americans are those personsliving in North America and having origins in any of the black racialgroups of Africa. In another example, self-reported persons of blackAfrican ancestry may have at least one parent of black African ancestryor at least one grandparent of black African ancestry.

The racial contribution in individual subjects may also be determined bygenetic analysis. Genetic analysis of ancestry may be carried out usingunlinked microsatellite markers such as those set out in Smith et al.(Am J Hum Genet 74, 1001-13 (2004)).

In certain embodiments, the invention relates to markers and/orhaplotypes identified in specific populations, as described in theabove. The person skilled in the art will appreciate that measures oflinkage disequilibrium (LD) may give different results when applied todifferent populations. This is due to different population history ofdifferent human populations as well as differential selective pressuresthat may have led to differences in LD in specific genomic regions. Itis also well known to the person skilled in the art that certainmarkers, e.g. SNP markers, are polymorphic in one population but not inanother. The person skilled in the art will however apply the methodsavailable and as taught ??herein to practice the present invention inany given human population. This may include assessment of polymorphicmarkers in the LD region of the present invention, so as to identifythose markers that give strongest association within the specificpopulation. Thus, the at-risk variants of the present invention mayreside on different haplotype background and in different frequencies invarious human populations. However, utilizing methods known in the artand the markers of the present invention, the invention can be practicedin any given human population.

Utility of Genetic Testing

The person skilled in the art will appreciate and understand that thevariants described herein in general do not, by themselves, provide anabsolute identification of individuals who will develop cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.The variants described herein do however indicate increased and/ordecreased likelihood that individuals carrying the at-risk or protectivevariants of the invention will develop symptoms associated with cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokeThis information is however extremely valuable in itself, as outlined inmore detail in the below, as it can be used to, for example, initiatepreventive measures at an early stage, perform regular physical and/ormental exams to monitor the progress and/or appearance of symptoms, orto schedule exams at a regular interval to identify the condition inquestion, so as to be able to apply treatment at an early stage.

The knowledge about a genetic variant that confers a risk of developingcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke offers the opportunity to apply a genetic test to distinguishbetween individuals with increased risk of developing the disease (i.e.carriers of the at-risk variant) and those with decreased risk ofdeveloping the disease (i.e. carriers of the protective variant). Thecore values of genetic testing, for individuals belonging to both of theabove mentioned groups, are the possibilities of being able to diagnosecardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke, or a predisposition to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke at an early stage andprovide information to the clinician about prognosis of cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokein order to be able to apply the most appropriate treatment.

Individuals with a family history of cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke and carriers of at-riskvariants may benefit from genetic testing since the knowledge of thepresence of a genetic risk factor, or evidence for increased risk ofbeing a carrier of one or more risk factors, may provide increasedincentive for implementing a healthier lifestyle, by avoiding orminimizing known environmental risk factors for cardiovascular diseasesrelated to cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke. Genetic testing of cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke patients mayfurthermore give valuable information about the primary cause of thedisease and can aid the clinician in selecting the best treatmentoptions and medication for each individual.

The present invention furthermore relates to risk assessment for cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,including determining whether an individual is at risk for developingcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke. The polymorphic markers of the present invention can be usedalone or in combination, as well as in combination with other factors,including other genetic risk factors or biomarkers, for risk assessmentof an individual for cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke. Many factors known to affect thepredisposition of an individual towards developing risk ofcardiovascular disease are susceptibility factors for cardiacarrhythmias (e.g., atrial fibrillation or atrial flutter) and/or stroke,and are known to the person skilled in the art and can be utilized insuch assessment. These include, but are not limited to, age, gender,smoking status, physical activity, waist-to-hip circumference ratio,family history of cardiac arrhythmia (in particular atrial fibrillationand/or atrial flutter) and/or stroke, previously diagnosed cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,obesity, hypertriglyceridemia, low HDL cholesterol, hypertension,elevated blood pressure, cholesterol levels, HDL cholesterol, LDLcholesterol, triglycerides, apolipoprotein AI and B levels, fibrinogen,ferritin, C-reactive protein and leukotriene levels. Particularbiomarkers that have been associated with Atrial fibrillation/Atrialflutter and stroke are discussed in Allard et al. (Clin Chem51:2043-2051 (2005) and Becker (J Thromb Thrombolys 19:71-75 (2005)).These include, but are not limited to, fibrin D-dimer, prothrombinactivation fragment 1.2 (F1.2), thrombin-antithrombin III complexes(TAT), fibrinopeptide A (FPA), lipoprotein-associated phospholipase A2(Ip-PLA2), beta-thromboglobulin, platelet factor 4, P-selectin, vonWillebrand Factor, pro-natriuretic peptide (BNP), matrixmetalloproteinase-9 (MMP-9), PARK7, nucleoside diphosphate kinase(NDKA), tau, neuron-specific enolase, B-type neurotrophic growth factor,astroglial protein S-100b, glial fibrillary acidic protein, C-reactiveprotein, seum amyloid A, marix metalloproteinase-9, vascular andintracellular cell adhesion molecules, tumor necrosis factor alpha, andinterleukins, including interleukin-1, -6, and -8). Circulatingprogenitor cells have also been implicated as being useful biomarkersfor AF. In particular embodiments, more than one biomarker is determinedfor an individual, and combined with results of a determination of atleast one polymorphic marker as described herein. Preferably, biomarkeris measured in plasma or serum from the individual. Alternatively, thebiomarker is determined in other suitable tissues containing measurableamounts of the biomarker, and such embodiments are also within scope ofthe invention.

Methods known in the art can be used for overall risk assessment,including multivariate analyses or logistic regression.

Atrial fibrillation is a disease of great significance both to theindividual patient and to the health care system as a whole. It can be apermanent condition but may also be paroxysmal and recurrent in whichcase it can be very challenging to diagnose. The most devastatingcomplication of atrial fibrillation and atrial flutter is the occurrenceof debilitating stroke. Importantly the risk of stroke is equal inpermanent and paroxysmal atrial fibrillation. It has repeatedly beenshown that therapy with warfarin anticoagulation can significantlyreduce the risk of first or further episodes of stroke in the setting ofatrial fibrillation. Therefor, anticoagulation with warfarin is standardtherapy for almost all patients with atrial fibrillation forstroke-prevention, whether they have the permanent or paroxysmal type.The only patients for whom warfarin is not strongly recommended arethose younger than 65 years old who are considered low-risk, i.e., theyhave no organic heart disease, including, neither hypertension nocoronary artery disease, no previous history of stroke or transientischemic attacks and no diabetes. This group has a lower risk of strokeand stroke-prevention with aspirin is recommended.

Due to the nature of paroxysmal atrial fibrillation it can be verydifficult to diagnose. When the patient seeks medical attention due todisease-related symptoms, such as palpitations, chest pain, shortness ofbreath, dizziness, heart failure, transient ischemic attacks or evenstroke, normal heart rhythm may already be restored precluding diagnosisof the arrhythmia. In these cases cardiac rhythm monitoring isfrequently applied in the attempt to diagnose the condition. The cardiacrhythm is commonly monitored continuously for 24 to 48 hours.Unfortunately atrial fibrillation episodes are unpredictable andfrequently missed by this approach. The opportunity to diagnose thearrhythmia, institute recommended therapy, and possibly prevent adebilitating first or recurrent stroke may be missed with devastatingresults to the patient. Prolonged and more complex cardiac rhythmmonitoring measures are available and applied occasionally when thesuspicion of atrial fibrillation is very strong. These tests areexpensive, the diagnostic yield with current approach is often low, andthey are used sparingly for this indication. In these circumstancesadditional risk stratification with genetic testing may be extremelyhelpful. Understanding that the individual in question carries either anat-risk or a protective genetic variant can be an invaluablecontribution to diagnostic and/or treatment decision making. This way,in some cases, unnecessary testing and therapy may be avoided, and inother cases, with the help of more aggressive diagnostic approach, thearrhythmia may be diagnosed and/or proper therapy initiated and latercomplications of disease diminished.

Methods of the Invention

Methods for risk assessment of cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke are described herein andare encompassed by the invention. The invention also encompasses methodsof assessing an individual for probability of response to a therapeuticagent for cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke, as well as methods for predicting theeffectiveness of a therapeutic agent to treat patients with cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.Kits for assaying a sample from a subject to detect susceptibility tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke are also encompassed by the invention.

Diagnostic and Screening Assays of the Invention

In certain embodiments, the present invention pertains to methods ofdiagnosing, or aiding in the diagnosis of, cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke or a susceptibilityto cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke, by detecting particular alleles at genetic markers thatappear more frequently in cardiac arrhythmia (e.g., atrial fibrillationor atrial flutter) and/or stroke subjects or subjects who aresusceptible to cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke. In a particular embodiment, the invention is amethod of diagnosing a susceptibility to cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke by detecting atleast one allele of at least one polymorphic marker (e.g., the markersdescribed herein). The present invention describes methods wherebydetection of particular alleles of particular markers or haplotypes isindicative of a susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. Such prognostic orpredictive assays can also be used to determine prophylactic treatmentof a subject prior to the onset of symptoms of cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke.

The present invention pertains in some embodiments to methods ofclinical applications of diagnosis, e.g., diagnosis performed by amedical professional, which may include an assessment or determinationof genetic risk variants, and their interpretation. In otherembodiments, the invention pertains to methods of risk assessment (ordiagnosis) performed by a layman or a non-medical professional. Recenttechnological advances in genotyping technologies, includinghigh-throughput genotyping of SNP markers, such as Molecular InversionProbe array technology (e.g., Affymetrix GeneChip), and BeadArrayTechnologies (e.g., Illumina GoldenGate and Infinium assays) have madeit possible for individuals to have their own genome assessed for largenumber of variations simultaneously, or up to one million SNPs. Theresulting genotype information, made available to the individual, can becompared to information from the public scientific literature aboutdisease or trait risk associated with various SNPs. The diagnosticapplication of disease-associated alleles as described herein, can thusbe performed either by a health professional based on results of aclinical test or by a layman, or non-medical professional, including anindividual providing service for performing an assessment of SNPsthrough SNP genotyping, either on an individual SNP basis or bylarge-scale high-throughput methods such as array technologies. In otherwords, the diagnosis or assessment of a susceptibility based on geneticrisk can be made by health professionals, genetic counselors, genotypeservices providers or by the layman, based on information about his/hergenotype and publications on various risk factors. In the presentcontext, the term “diagnosing”, and “diagnose a susceptibility”, ismeant to refer to any available diagnostic method, including thosementioned above.

In addition, in certain other embodiments, the present inventionpertains to methods of diagnosing, or aiding in the diagnosis of, adecreased susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke by detecting particulargenetic marker alleles or haplotypes that appear less frequently incardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke patients than in individual not diagnosed with cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke or in thegeneral population.

As described and exemplified herein, particular marker alleles orhaplotypes (e.g. the markers and haplotypes as listed in Table 5 (Tables5A and 5B) and markers in linkage disequilibrium therewith, e.g., themarkers listed in Tables 4 and/or 9 markers in linkage disequilibriumtherewith, e.g., the markers as set forth in Table 19) are associatedwith cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke. In one embodiment, the marker allele or haplotype is onethat confers a significant risk or susceptibility to cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke. In anotherembodiment, the invention relates to a method of diagnosing asusceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke in a human individual, the methodcomprising determining the presence or absence of at least one allele ofat least one polymorphic marker in a nucleic acid sample obtained fromthe individual, wherein the at least one polymorphic marker is selectedfrom the group consisting of the polymorphic markers listed in Tables 5Aand 5B, and markers in linkage disequilibrium therewith. In anotherembodiment, the invention pertains to methods of diagnosing asusceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke in a human individual, by screening for atleast one marker allele or haplotype as listed in Tables 5A and 5B ormarkers in linkage disequilibrium therewith. In another embodiment, themarker allele or haplotype is more frequently present in a subjecthaving, or who is susceptible to, cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke (affected), as compared tothe frequency of its presence in a healthy subject (control, such aspopulation controls). In certain embodiments, the significance ofassociation of the at least one marker allele or haplotype ischaracterized by a p value<0.05. In other embodiments, the significanceof association is characterized by smaller p-values, such as <0.01,<0.001, <0.0001, <0.00001, <0.000001, <0.0000001, <0.00000001 or<0.000000001.

In these embodiments, the presence of the at least one marker allele orhaplotype is indicative of a susceptibility to cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke. These diagnosticmethods involve detecting the presence or absence of at least one markerallele or haplotype that is associated with cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke. The haplotypesdescribed herein include combinations of alleles at various geneticmarkers (e.g., SNPs, microsatellites). The detection of the particulargenetic marker alleles that make up the particular haplotypes can beperformed by a variety of methods described herein and/or known in theart. For example, genetic markers can be detected at the nucleic acidlevel (e.g., by direct nucleotide sequencing or by other means known tothe skilled in the art) or at the amino acid level if the genetic markeraffects the coding sequence of a protein encoded by a cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke-associatednucleic acid (e.g., by protein sequencing or by immunoassays usingantibodies that recognize such a protein). The marker alleles orhaplotypes of the present invention correspond to fragments of a genomicDNA sequence associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. Such fragments encompassthe DNA sequence of the polymorphic marker or haplotype in question, butmay also include DNA segments in strong LD (linkage disequilibrium) withthe marker or haplotype. In one embodiment, such segments comprisessegments in LD with the marker or haplotype as determined by a value ofr² greater than 0.2 and/or |D′|>0.8.

In one embodiment, diagnosis of a susceptibility to cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke can beaccomplished using hybridization methods, such as Southern analysis,Northern analysis, and/or in situ hybridizations (see Current Protocolsin Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons,including all supplements). A biological sample from a test subject orindividual (a “test sample”) of genomic DNA, RNA, or cDNA is obtainedfrom a subject suspected of having, being susceptible to, or predisposedfor cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke (the “test subject”). The subject can be an adult, child,or fetus. The test sample can be from any source that contains genomicDNA, such as a blood sample, sample of amniotic fluid, sample ofcerebrospinal fluid, or tissue sample from skin, muscle, buccal orconjunctival mucosa, placenta, gastrointestinal tract or other organs. Atest sample of DNA from fetal cells or tissue can be obtained byappropriate methods, such as by amniocentesis or chorionic villussampling. The DNA, RNA, or cDNA sample is then examined. The presence ofa specific marker allele can be indicated by sequence-specifichybridization of a nucleic acid probe specific for the particularallele. The presence of more than specific marker allele or a specifichaplotype can be indicated by using several sequence-specific nucleicacid probes, each being specific for a particular allele. In oneembodiment, a haplotype can be indicated by a single nucleic acid probethat is specific for the specific haplotype (i.e., hybridizesspecifically to a DNA strand comprising the specific marker allelescharacteristic of the haplotype). A sequence-specific probe can bedirected to hybridize to genomic DNA, RNA, or cDNA. A “nucleic acidprobe”, as used herein, can be a DNA probe or an RNA probe thathybridizes to a complementary sequence. One of skill in the art wouldknow how to design such a probe so that sequence specific hybridizationwill occur only if a particular allele is present in a genomic sequencefrom a test sample.

To diagnose a susceptibility to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke, a hybridization sample isformed by contacting the test sample containing an atrial fibrillationand/or stroke-associated nucleic acid, such as a genomic DNA sample,with at least one nucleic acid probe. A non-limiting example of a probefor detecting mRNA or genomic DNA is a labeled nucleic acid probe thatis capable of hybridizing to mRNA or genomic DNA sequences describedherein. The nucleic acid probe can be, for example, a full-lengthnucleic acid molecule, or a portion thereof, such as an oligonucleotideof at least 15, 30, 50, 100, 250 or 500 nucleotides in length that issufficient to specifically hybridize under stringent conditions toappropriate mRNA or genomic DNA. The nucleotide acid probe may be up to1000 or more nucleotides in length, including up to 500 nucleotides, 400nucleotide, 300 nucleotides, 200 nucleotides or 100 nucleotides. Certainembodiments include nucleotide probes that are from 15 to 1000nucleotides in length. Other embodiments pertain to use of nucleotideprobes that are from 15 to 500 nucleotides in length, or from 15 to 400nucleotides in length, or from 20 to 400 nucleotides in length. Othersize ranges of the nucleotide probes of the invention are contemplated,as well known to the skilled person. In one embodiment, the nucleic acidprobe can comprise all or a portion of the nucleotide sequence of LDBlock C04, as described herein, optionally comprising at least oneallele of a marker described herein, or at least one haplotype describedherein, or the probe can be the complementary sequence of such asequence. In a particular embodiment, the nucleic acid probe is aportion of the nucleotide sequence of LD Block C04 as set forth in SEQID NO:50 or, as described herein, optionally comprising at least oneallele of a marker described herein, or at least one allele of onepolymorphic marker or haplotype comprising at least one polymorphicmarker described herein, or the probe can be the complementary sequenceof such a sequence. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization can beperformed by methods well known to the person skilled in the art (see,e.g., Current Protocols in Molecular Biology, Ausubel, F. et al., eds.,John Wiley & Sons, including all supplements). In one embodiment,hybridization refers to specific hybridization, i.e., hybridization withno mismatches (exact hybridization). In one embodiment, thehybridization conditions for specific hybridization are high stringency.

Specific hybridization, if present, is detected using standard methods.If specific hybridization occurs between the nucleic acid probe and thenucleic acid in the test sample, then the sample contains the allelethat is complementary to the nucleotide that is present in the nucleicacid probe. The process can be repeated for any markers of the presentinvention, or markers that make up a haplotype of the present invention,or multiple probes can be used concurrently to detect more than onemarker alleles at a time. It is also possible to design a single probecontaining more than one marker alleles of a particular haplotype (e.g.,a probe containing alleles complementary to 2, 3, 4, 5 or all of themarkers that make up a particular haplotype). Detection of theparticular markers of the haplotype in the sample is indicative that thesource of the sample has the particular haplotype (e.g., a haplotype)and therefore is susceptible to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke.

In another hybridization method, Northern analysis (see CurrentProtocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley &Sons, supra) is used to identify the presence of a polymorphismassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke. For Northern analysis, a test sample of RNA isobtained from the subject by appropriate means. As described herein,specific hybridization of a nucleic acid probe to RNA from the subjectis indicative of a particular allele complementary to the probe. Forrepresentative examples of use of nucleic acid probes, see, for example,U.S. Pat. Nos. 5,288,611 and 4,851,330.

Additionally, or alternatively, a peptide nucleic acid (PNA) probe canbe used in addition to, or instead of, a nucleic acid probe in thehybridization methods described herein. A PNA is a DNA mimic having apeptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units,with an organic base (A, G, C, T or U) attached to the glycine nitrogenvia a methylene carbonyl linker (see, for example, Nielsen, P., et al.,Bioconjug. Chem. 5:3-7 (1994)). The PNA probe can be designed tospecifically hybridize to a molecule in a sample suspected of containingone or more of the marker alleles or haplotypes that are associated withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke. Hybridization of the PNA probe is thus diagnostic for cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokeor a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke.

In one embodiment of the invention, a test sample containing genomic DNAobtained from the subject is collected and the polymerase chain reaction(PCR) is used to amplify a fragment comprising one or more markers orhaplotypes of the present invention. As described herein, identificationof a particular marker allele or haplotype associated with cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,can be accomplished using a variety of methods (e.g., sequence analysis,analysis by restriction digestion, specific hybridization, singlestranded conformation polymorphism assays (SSCP), electrophoreticanalysis, etc.). In another embodiment, diagnosis is accomplished byexpression analysis using quantitative PCR (kinetic thermal cycling).This technique can, for example, utilize commercially availabletechnologies, such as TaqMan® (Applied Biosystems, Foster City, Calif.).The technique can assess the presence of an alteration in the expressionor composition of a polypeptide or spiking variant(s) that is encoded bya nucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. Further, the expressionof the variant(s) can be quantified as physically or functionallydifferent.

In another method of the invention, analysis by restriction digestioncan be used to detect a particular allele if the allele results in thecreation or elimination of a restriction site relative to a referencesequence. Restriction fragment length polymorphism (RFLP) analysis canbe conducted, e.g., as described in Current Protocols in MolecularBiology, supra. The digestion pattern of the relevant DNA fragmentindicates the presence or absence of the particular allele in thesample.

Sequence analysis can also be used to detect specific alleles orhaplotypes associated with cardiac arrhythmia (e.g., atrial fibrillationor atrial flutter) and/or stroke (e.g. the polymorphic markers of Table5 (Tables 5A and 5B), Table 9 and/or Table 19). Therefore, in oneembodiment, determination of the presence or absence of a particularmarker alleles or haplotypes comprises sequence analysis of a testsample of DNA or RNA obtained from a subject or individual. PCR or otherappropriate methods can be used to amplify a portion of a nucleic acidassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke, and the presence of a specific allele can thenbe detected directly by sequencing the polymorphic site (or multiplepolymorphic sites in a haplotype) of the genomic DNA in the sample.

Allele-specific oligonucleotides can also be used to detect the presenceof a particular allele in a nucleic acid associated with cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,(e.g. the polymorphic markers of Table 5 (Tables 5A and 5B), Table 9and/or Table 19), through the use of dot-blot hybridization of amplifiedoligonucleotides with allele-specific oligonucleotide (ASO) probes (see,for example, Saiki, R. et al., Nature, 324: 163-166 (1986)). An“allele-specific oligonucleotide” (also referred to herein as an“allele-specific oligonucleotide probe”) is an oligonucleotide ofapproximately 10-500 base pairs, approximately 15-400 base pairs,approximately 15-200 base pairs, approximately 15-100 base pairs,approximately 15-50 base pairs, or approximately 15-30 base pairs, thatspecifically hybridizes to a nucleic acid associated with cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke,and which contains a specific allele at a polymorphic site (e.g., amarker or haplotype as described herein). An allele-specificoligonucleotide probe that is specific for one or more particular anucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke can be prepared usingstandard methods (see, e.g., Current Protocols in Molecular Biology,supra). PCR can be used to amplify the desired region. The DNAcontaining the amplified region can be dot-blotted using standardmethods (see, e.g., Current Protocols in Molecular Biology, supra), andthe blot can be contacted with the oligonucleotide probe. The presenceof specific hybridization of the probe to the amplified region can thenbe detected. Specific hybridization of an allele-specificoligonucleotide probe to DNA from the subject is indicative of aspecific allele at a polymorphic site associated with cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke (see, e.g.,Gibbs, R. et al., Nucleic Acids Res., 17:2437-2448 (1989) and WO93/22456).

With the addition of such analogs as locked nucleic acids (LNAs), thesize of primers and probes can be reduced to as few as 8 bases. LNAs area novel class of bicyclic DNA analogs in which the 2′ and 4′ positionsin the furanose ring are joined via an O-methylene (oxy-LNA),S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety. Common toall of these LNA variants is an affinity toward complementary nucleicacids, which is by far the highest reported for a DNA analog. Forexample, particular all oxy-LNA nonamers have been shown to have meltingtemperatures (T_(m)) of 64° C. and 74° C. when in complex withcomplementary DNA or RNA, respectively, as opposed to 28° C. for bothDNA and RNA for the corresponding DNA nonamer. Substantial increases inT_(m) are also obtained when LNA monomers are used in combination withstandard DNA or RNA monomers. For primers and probes, depending on wherethe LNA monomers are included (e.g., the 3′ end, the 5′ end, or in themiddle), the T_(m) could be increased considerably.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequence segments from a subject,can be used to identify polymorphisms in a nucleic acid associated withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke (e.g. the polymorphic markers of Tables 5A and 5B and markers inlinkage disequilibrium therewith). For example, an oligonucleotide arraycan be used. Oligonucleotide arrays typically comprise a plurality ofdifferent oligonucleotide probes that are coupled to a surface of asubstrate in different known locations. These oligonucleotide arrays,also described as “Genechips™,” have been generally described in the art(see, e.g., U.S. Pat. No. 5,143,854, PCT Patent Publication Nos. WO90/15070 and 92/10092). These arrays can generally be produced usingmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phaseoligonucleotide synthesis methods, or by other methods known to theperson skilled in the art (see, e.g., Fodor, S. et al., Science,251:767-773 (1991); Pirrung et al., U.S. Pat. No. 5,143,854 (see alsopublished PCT Application No. WO 90/15070); and Fodor. S. et al.,published PCT Application No. WO 92/10092 and U.S. Pat. No. 5,424,186,the entire teachings of each of which are incorporated by referenceherein). Techniques for the synthesis of these arrays using mechanicalsynthesis methods are described in, e.g., U.S. Pat. No. 5,384,261; theentire teachings of which are incorporated by reference herein. Inanother example, linear arrays can be utilized. Additional descriptionsof use of oligonucleotide arrays for detection of polymorphisms can befound, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832, theentire teachings of both of which are incorporated by reference herein.

Other methods of nucleic acid analysis that are available to thoseskilled in the art can be used to detect a particular allele at apolymorphic site associated with atrial fibrillation and/or stroke (e.g.the polymorphic markers of Table 5 (Tables 5A and 5B), Table 9 and/orTable 19). Representative methods include, for example, direct manualsequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81:1991-1995 (1988); Sanger, F., et al., Proc. Natl. Acad. Sci. USA,74:5463-5467 (1977); Beavis, et al., U.S. Pat. No. 5,288,644); automatedfluorescent sequencing; single-stranded conformation polymorphism assays(SSCP); clamped denaturing gel electrophoresis (CDGE); denaturinggradient gel electrophoresis (DGGE) (Sheffield, V., et al., Proc. Natl.Acad. Sci. USA, 86:232-236 (1989)), mobility shift analysis (Orita, M.,et al., Proc. Natl. Acad. Sci. USA, 86:2766-2770 (1989)), restrictionenzyme analysis (Flavell, R., et al., Cell, 15:25-41 (1978); Geever, R.,et al., Proc. Natl. Acad. Sci. USA, 78:5081-5085 (1981)); heteroduplexanalysis; chemical mismatch cleavage (CMC) (Cotton, R., et al., Proc.Natl. Acad. Sci. USA, 85:4397-4401 (1985)); RNase protection assays(Myers, R., et al., Science, 230:1242-1246 (1985); use of polypeptidesthat recognize nucleotide mismatches, such as E. coli mutS protein; andallele-specific PCR.

In another embodiment of the invention, diagnosis of cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke or asusceptibility to cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke can be made by examining expression and/orcomposition of a polypeptide encoded by a nucleic acid associated withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke in those instances where the genetic marker(s) or haplotype(s) ofthe present invention result in a change in the composition orexpression of the polypeptide. Thus, diagnosis of a susceptibility tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke can be made by examining expression and/or composition of one ofthese polypeptides, or another polypeptide encoded by a nucleic acidassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke, in those instances where the genetic marker orhaplotype of the present invention results in a change in thecomposition or expression of the polypeptide. The haplotypes and markersof the present invention that show association to cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke may play arole through their effect on one or more of these nearby genes (e.g.,the PITX2 gene). Possible mechanisms affecting these genes include,e.g., effects on transcription, effects on RNA splicing, alterations inrelative amounts of alternative splice forms of mRNA, effects on RNAstability, effects on transport from the nucleus to cytoplasm, andeffects on the efficiency and accuracy of translation.

Thus, in another embodiment, the variants (markers or haplotypes) of theinvention showing association to cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke affect the expression of anearby gene. It is well known that regulatory element affecting geneexpression may be located tenths or even hundreds of kilobases away fromthe promoter region of a gene. By assaying for the presence or absenceof at least one allele of at least one polymorphic marker of the presentinvention, it is thus possible to assess the expression level of suchnearby genes. It is thus contemplated that the detection of the markersor haplotypes of the present invention can be used for assessingexpression for one or more genes that are linked to cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke.

A variety of methods can be used for detecting protein expressionlevels, including enzyme linked immunosorbent assays (ELISA), Westernblots, immunoprecipitations and immunofluorescence. A test sample from asubject is assessed for the presence of an alteration in the expressionand/or an alteration in composition of the polypeptide encoded by anucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. An alteration inexpression of a polypeptide encoded by a nucleic acid associated withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke can be, for example, an alteration in the quantitativepolypeptide expression (i.e., the amount of polypeptide produced). Analteration in the composition of a polypeptide encoded by a nucleic acidassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke is an alteration in the qualitative polypeptideexpression (e.g., expression of a mutant polypeptide or of a differentsplicing variant). In one embodiment, diagnosis of a susceptibility tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke is made by detecting a particular splicing variant encoded by anucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke, or a particular patternof splicing variants.

Both such alterations (quantitative and qualitative) can also bepresent. An “alteration” in the polypeptide expression or composition,as used herein, refers to an alteration in expression or composition ina test sample, as compared to the expression or composition of thepolypeptide in a control sample. A control sample is a sample thatcorresponds to the test sample (e.g., is from the same type of cells),and is from a subject who is not affected by, and/or who does not have asusceptibility to, cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke. In one embodiment, the control sample isfrom a subject that does not possess a marker allele or haplotype asdescribed herein. Similarly, the presence of one or more differentsplicing variants in the test sample, or the presence of significantlydifferent amounts of different splicing variants in the test sample, ascompared with the control sample, can be indicative of a susceptibilityto cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke. An alteration in the expression or composition of thepolypeptide in the test sample, as compared with the control sample, canbe indicative of a specific allele in the instance where the allelealters a splice site relative to the reference in the control sample.Various means of examining expression or composition of a polypeptideencoded by a nucleic acid are known to the person skilled in the art andcan be used, including spectroscopy, colorimetry, electrophoresis,isoelectric focusing, and immunoassays (e.g., David et al., U.S. Pat.No. 4,376,110) such as immunoblotting (see, e.g., Current Protocols inMolecular Biology, particularly chapter 10, supra).

For example, in one embodiment, an antibody (e.g., an antibody with adetectable label) that is capable of binding to a polypeptide encoded bya nucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke can be used. Antibodiescan be polyclonal or monoclonal. An intact antibody, or a fragmentthereof (e.g., Fv, Fab, Fab′, F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a labeled secondary antibody(e.g., a fluorescently-labeled secondary antibody) and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin.

In one embodiment of this method, the level or amount of polypeptideencoded by a nucleic acid associated with cardiac arrhythmia (e.g.,atrial fibrillation or atrial flutter) and/or stroke in a test sample iscompared with the level or amount of the polypeptide in a controlsample. A level or amount of the polypeptide in the test sample that ishigher or lower than the level or amount of the polypeptide in thecontrol sample, such that the difference is statistically significant,is indicative of an alteration in the expression of the polypeptideencoded by the nucleic acid, and is diagnostic for a particular alleleor haplotype responsible for causing the difference in expression.Alternatively, the composition of the polypeptide in a test sample iscompared with the composition of the polypeptide in a control sample. Inanother embodiment, both the level or amount and the composition of thepolypeptide can be assessed in the test sample and in the controlsample.

In another embodiment, the diagnosis of a susceptibility to cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokeis made by detecting at least one marker or haplotypes of the presentinvention (e.g., associated alleles of the markers listed in Tables 5Aand 5B, and markers in linkage disequilibrium therewith), in combinationwith an additional protein-based, RNA-based or DNA-based assay. Themethods of the invention can also be used in combination with ananalysis of a subject's family history and risk factors (e.g.,environmental risk factors, lifestyle risk factors).

Kits

Kits useful in the methods and procedures of the invention comprisecomponents useful in any of the methods described herein, including forexample, hybridization probes, restriction enzymes (e.g., for RFLPanalysis), allele-specific oligonucleotides, antibodies that bind to analtered polypeptide encoded by a nucleic acid of the invention asdescribed herein (e.g., a genomic segment comprising at least onepolymorphic marker and/or haplotype of the present invention) or to anon-altered (native) polypeptide encoded by a nucleic acid of theinvention as described herein, means for amplification of a nucleic acidassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke, means for analyzing the nucleic acid sequence ofa nucleic acid associated with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke, means for analyzing theamino acid sequence of a polypeptide encoded by a nucleic acidassociated with cardiac arrhythmia (e.g., atrial fibrillation or atrialflutter) and/or stroke, etc. The kits can for example include necessarybuffers, nucleic acid primers for amplifying nucleic acids of theinvention (e.g., one or more of the polymorphic markers as describedherein), and reagents for allele-specific detection of the fragmentsamplified using such primers and necessary enzymes (e.g., DNApolymerase). Additionally, kits can provide reagents for assays to beused in combination with the methods of the present invention, e.g.,reagents for use with cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke diagnostic assays.

In one embodiment, the invention is a kit for assaying a sample from asubject to detect the presence of cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke or a susceptibility tocardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke in a subject, wherein the kit comprises reagents necessary forselectively detecting at least one allele of at least one polymorphismof the present invention in the genome of the individual. In aparticular embodiment, the reagents comprise at least one contiguousoligonucleotide that hybridizes to a fragment of the genome of theindividual comprising at least one polymorphism of the presentinvention. In another embodiment, the reagents comprise at least onepair of oligonucleotides that hybridize to opposite strands of a genomicsegment obtained from a subject, wherein each oligonucleotide primerpair is designed to selectively amplify a fragment of the genome of theindividual that includes at least one polymorphism, wherein thepolymorphism is selected from the group consisting of the polymorphismsas listed in Tables 5A and 5B and polymorphic markers in linkagedisequilibrium therewith. In yet another embodiment the fragment is atleast 20 base pairs in size. Such oligonucleotides or nucleic acids(e.g., oligonucleotide primers) can be designed using portions of thenucleic acid sequence flanking polymorphisms (e.g., SNPs ormicrosatellites) that are indicative of cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. In another embodiment,the kit comprises one or more labeled nucleic acids capable ofallele-specific detection of one or more specific polymorphic markers orhaplotypes associated with cardiac arrhythmia (e.g., atrial fibrillationor atrial flutter) and/or stroke, and reagents for detection of thelabel. Suitable labels include, e.g., a radioisotope, a fluorescentlabel, an enzyme label, an enzyme co-factor label, a magnetic label, aspin label, an epitope label.

In particular embodiments, the polymorphic marker or haplotype to bedetected by the reagents of the kit comprises one or more markers, twoor more markers, three or more markers, four or more markers or five ormore markers selected from the group consisting of the markers in Tables5A and 5B. In another embodiment, the marker or haplotype to be detectedcomprises the markers listed in Tables 5A and 5B. In another embodiment,the marker or haplotype to be detected comprises the markers listed inTables 4 and 9. In another embodiment, the marker or haplotype to bedetected comprises at least one marker from the group of markers instrong linkage disequilibrium, as defined by values of r² greater than0.2, to at least one of the group of markers consisting of the markerslisted in Tables 5A and 5B. In another embodiment, the marker orhaplotype to be detected comprises at least one marker from the markersin strong linkage disequilibrium, as defined by values of r² greaterthan 0.2, to at least one of the group of markers consisting of themarkers listed in Tables 4 and 9. In another embodiment, the marker orhaplotype to be detected comprises marker rs2220427 (SEQ ID NO:1) ormarker rs1033464 (SEQ ID NO:41), or markers in linkage disequilibriumtherewith. In another embodiment, the marker or haplotype to be detectedcomprises at least one of the markers set forth in Table 19. In anotherembodiment, the marker or haplotype to be detected comprises markersD4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ IDNO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), andrs13143308 (SEQ ID NO:51) and markers in linkage disequilibriumtherewith. In yet another embodiment, the marker or haplotype comprisesthe at-risk alleles-2, -4 and/or -8 in marker D4S406, allele A of markerrs2634073, allele T of marker rs2200733, allele T of marker rs2220427,allele T of marker rs10033464, and/or allele G of marker rs13143308. Inone such embodiment, linkage disequilibrium is defined by values of r²greater than 0.1. In another such embodiment, linkage disequilibrium isdefined by values of r² greater than 0.2.

In one preferred embodiment, the kit for detecting the markers of theinvention comprises a detection oligonucleotide probe, that hybridizesto a segment of template DNA containing a SNP polymorphisms to bedetected, an enhancer oligonucleotide probe and an endonuclease. Asexplained in the above, the detection oligonucleotide probe comprises afluorescent moiety or group at its 3′ terminus and a quencher at its 5°terminus, and an enhancer oligonucleotide, is employed, as described byKutyavin et al. (Nucleic Acid Res. 34:e128 (2006)). The fluorescentmoiety can be Gig Harbor Green or Yakima Yellow, or other suitablefluorescent moieties. The detection probe is designed to hybridize to ashort nucleotide sequence that includes the SNP polymorphism to bedetected. Preferably, the SNP is anywhere from the terminal residue to-6 residues from the 3′ end of the detection probe. The enhancer is ashort oligonucleotide probe which hybridizes to the DNA template 3′relative to the detection probe. The probes are designed such that asingle nucleotide gap exists between the detection probe and theenhancer nucleotide probe when both are bound to the template. The gapcreates a synthetic abasic site that is recognized by an endonuclease,such as Endonuclease IV. The enzyme cleaves the dye off the fullycomplementary detection probe, but cannot cleave a detection probecontaining a mismatch. Thus, by measuring the fluorescence of thereleased fluorescent moiety, assessment of the presence of a particularallele defined by nucleotide sequence of the detection probe can beperformed.

The detection probe can be of any suitable size, although preferably theprobe is relatively short. In one embodiment, the probe is from 5-100nucleotides in length. In another embodiment, the probe is from 10-50nucleotides in length, and in another embodiment, the probe is from12-30 nucleotides in length. Other lengths of the probe are possible andwithin scope of the skill of the average person skilled in the art.

In a preferred embodiment, the DNA template containing the SNPpolymorphism is amplified by Polymerase Chain Reaction (PCR) prior todetection, and primers for such amplification are included in thereagent kit. In such an embodiment, the amplified DNA serves as thetemplate for the detection probe and the enhancer probe.

Certain embodiments of the detection probe, the enhancer probe, and/orthe primers used for amplification of the template by PCR include theuse of modified bases, including modified A and modified G. The use ofmodified bases can be useful for adjusting the melting temperature ofthe nucleotide molecule (probe and/or primer) to the template DNA, forexample for increasing the melting temperature in regions containing alow percentage of G or C bases, in which modified A with the capabilityof forming three hydrogen bonds to its complementary T can be used, orfor decreasing the melting temperature in regions containing a highpercentage of G or C bases, for example by using modified G bases thatform only two hydrogen bonds to their complementary C base in a doublestranded DNA molecule. In a preferred embodiment, modified bases areused in the design of the detection nucleotide probe. Any modified baseknown to the skilled person can be selected in these methods, and theselection of suitable bases is well within the scope of the skilledperson based on the teachings herein and known bases available fromcommercial sources as known to the skilled person.

In one of such embodiments, the presence of the marker or haplotype isindicative of a susceptibility (increased susceptibility or decreasedsusceptibility) to atrial fibrillation and/or stroke. In anotherembodiment, the presence of the marker or haplotype is indicative ofresponse to atrial fibrillation and/or stroke therapeutic agent. Inanother embodiment, the presence of the marker or haplotype isindicative of atrial fibrillation and/or stroke prognosis. In yetanother embodiment, the presence of the marker or haplotype isindicative of progress of atrial fibrillation and/or stroke treatment.Such treatment may include intervention by surgery, medication or byother means (e.g., lifestyle changes).

Therapeutic Agents

Variants of the present invention (e.g., the markers and/or haplotypesof the invention, e.g., the markers listed in Tables 5A and 5B and/orTable 19) can be used to identify novel therapeutic targets for cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.For example, genes containing, or in linkage disequilibrium with,variants (markers and/or haplotypes) associated with cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke, or theirproducts, as well as genes or their products that are directly orindirectly regulated by or interact with these variant genes or theirproducts, can be targeted for the development of therapeutic agents totreat cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke, or prevent or delay onset of symptoms associated withcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke. Therapeutic agents may comprise one or more of, for example,small non-protein and non-nucleic acid molecules, proteins, peptides,protein fragments, nucleic acids (DNA, RNA), PNA (peptide nucleicacids), or their derivatives or mimetics which can modulate the functionand/or levels of the target genes or their gene products.

The nucleic acids and/or variants of the invention, or nucleic acidscomprising their complementary sequence, may be used as antisenseconstructs to control gene expression in cells, tissues or organs. Themethodology associated with antisense techniques is well known to theskilled artisan, and is described and reviewed in Antisense DrugTechnology: Principles, Strategies, and Applications, Crooke, ed.,Marcel Dekker Inc., New York (2001). In general, antisense nucleic acidmolecules are designed to be complementary to a region of mRNA expressedby a gene, so that the antisense molecule hybridizes to the mRNA, thusblocking translation of the mRNA into protein. Several classes ofantisense oligonucleotide are known to those skilled in the art,including cleavers and blockers. The former bind to target RNA sites,activate intracellular nucleases (e.g., RnaseH or Rnase L), that cleavethe target RNA. Blockers bind to target RNA, inhibit protein translationby steric hindrance of the ribosomes. Examples of blockers includenucleic acids, morpholino compounds, locked nucleic acids andmethylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002)).Antisense oligonucleotides are useful directly as therapeutic agents,and are also useful for determining and validating gene function, forexample by gene knock-out or gene knock-down experiments. Antisensetechnology is further described in Layery et al., Curr. Opin. DrugDiscov. Devel. 6:561-569 (2003), Stephens et al., Curr. Opin. Mol. Ther.5:118-122 (2003), Kurreck, Eur. J. Biochem. 270:1628-44 (2003), Dias etal., Mol. Cancer Ter. 1:347-55 (2002), Chen, Methods Mol. Med.75:621-636 (2003), Wang et al., Curr. Cancer Drug Targets 1:177-96(2001), and Bennett, Antisense Nucleic Acid Drug. Dev. 12:215-24 (2002)

The variants described herein can be used for the selection and designof antisense reagents that are specific for particular variants. Usinginformation about the variants described herein, antisenseoligonucleotides or other antisense molecules that specifically targetmRNA molecules that contain one or more variants of the invention can bedesigned. In this manner, expression of mRNA molecules that contain oneor more variant of the present invention (markers and/or haplotypes) canbe inhibited or blocked. In one embodiment, the antisense molecules aredesigned to specifically bind a particular allelic form (i.e., one orseveral variants (alleles and/or haplotypes)) of the target nucleicacid, thereby inhibiting translation of a product originating from thisspecific allele or haplotype, but which do not bind other or alternatevariants at the specific polymorphic sites of the target nucleic acidmolecule.

As antisense molecules can be used to inactivate mRNA so as to inhibitgene expression, and thus protein expression, the molecules can be usedto treat a disease or disorder, such as cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. The methodology caninvolve cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Such mRNA regions include, forexample, protein-coding regions, in particular protein-coding regionscorresponding to catalytic activity, substrate and/or ligand bindingsites, or other functional domains of a protein.

The phenomenon of RNA interference (RNAi) has been actively studied forthe last decade, since its original discovery in C. elegans (Fire etal., Nature 391:806-11 (1998)), and in recent years its potential use intreatment of human disease has been actively pursued (reviewed in Kim &Rossi, Nature Rev. Genet. 8:173-204 (2007)). RNA interference (RNAi),also called gene silencing, is based on using double-stranded RNAmolecules (dsRNA) to turn off specific genes. In the cell, cytoplasmicdouble-stranded RNA molecules (dsRNA) are processed by cellularcomplexes into small interfering RNA (siRNA). The siRNA guide thetargeting of a protein-RNA complex to specific sites on a target mRNA,leading to cleavage of the mRNA (Thompson, Drug Discovery Today,7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or23 nucleotides in length. Thus, one aspect of the invention relates toisolated nucleic acid molecules, and the use of those molecules for RNAinterference, i.e. as small interfering RNA molecules (siRNA). In oneembodiment, the isolated nucleic acid molecules are 18-26 nucleotides inlength, preferably 19-25 nucleotides in length, more preferably 20-24nucleotides in length, and more preferably 21, 22 or 23 nucleotides inlength.

Another pathway for RNAi-mediated gene silencing originates inendogenously encoded primary microRNA (pri-miRNA) transcripts, which areprocessed in the cell to generate precursor miRNA (pre-miRNA). ThesemiRNA molecules are exported from the nucleus to the cytoplasm, wherethey undergo processing to generate mature miRNA molecules (miRNA),which direct translational inhibition by recognizing target sites in the3′ untranslated regions of mRNAs, and subsequent mRNA degradation byprocessing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet.8:173-204 (2007)).

Clinical applications of RNAi include the incorporation of syntheticsiRNA duplexes, which preferably are approximately 20-23 nucleotides insize, and preferably have 3′ overlaps of 2 nucleotides. Knockdown ofgene expression is established by sequence-specific design for thetarget mRNA. Several commercial sites for optimal design and synthesisof such molecules are known to those skilled in the art.

Other applications provide longer siRNA molecules (typically 25-30nucleotides in length, preferably about 27 nucleotides), as well assmall hairpin RNAs (shRNAs; typically about 29 nucleotides in length).The latter are naturally expressed, as described in Amarzguioui et al.(FEBS Lett. 579:5974-81 (2005)). Chemically synthetic siRNAs and shRNAsare substrates for in vivo processing, and in some cases provide morepotent gene-silencing than shorter designs (Kim et al., NatureBiotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol.23:227-231 (2005)). In general siRNAs provide for transient silencing ofgene expression, because their intracellular concentration is diluted bysubsequent cell divisions. By contrast, expressed shRNAs mediatelong-term, stable knockdown of target transcrips, for as long astranscription of the shRNA takes place (Marques et al., NatureBiotechnol. 23:559-565 (2006); Brummelkamp et al., Science 296: 550-553(2002)).

Since RNAi molecules, including siRNA, miRNA and shRNA, act in asequence-dependent manner, the variants of the present invention (e.g.,the markers and haplotypes associated with LD block C04, e.g., themarkers listed in Tables 5A and 5B) can be used to design RNAi reagentsthat recognize specific nucleic acid molecules comprising specificalleles and/or haplotypes (e.g., the alleles and/or haplotypes of thepresent invention), while not recognizing nucleic acid moleculescomprising other alleles or haplotypes. These RNAi reagents can thusrecognize and destroy the target nucleic acid molecules. As withantisense reagents, RNAi reagents can be useful as therapeutic agents(i.e., for turning off disease-associated genes or disease-associatedgene variants), but may also be useful for characterizing and validatinggene function (e.g., by gene knock-out or gene knock-down experiments).

Delivery of RNAi may be performed by a range of methodologies known tothose skilled in the art. Methods utilizing non-viral delivery includecholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chainantibody fragment (Fab), aptamers and nanoparticles. Viral deliverymethods include use of lentivirus, adenovirus and adeno-associatedvirus. The siRNA molecules are in some embodiments chemically modifiedto increase their stability. This can include modifications at the 2′position of the ribose, including 2′-O-methylpurines and2′-fluoropyrimidines, which provide resistance to Rnase activity. Otherchemical modifications are possible and known to those skilled in theart.

The following references provide a further summary of RNAi, andpossibilities for targeting specific genes using RNAi: Kim & Rossi, Nat.Rev. Genet. 8:173-184 (2007), Chen & Rajewsky, Nat. Rev. Genet. 8:93-103 (2007), Reynolds, et al., Nat. Biotechnol. 22:326-330 (2004), Chiet al., Proc. Natl. Acad. Sci. USA 100:6343-6346 (2003), Vickers et al.,J. Biol. Chem. 278:7108-7118 (2003), Agami, Curr. Opin. Chem. Biol.6:829-834 (2002), Layery, et al., Curr. Opin. Drug Discov. Devel.6:561-569 (2003), Shi, Trends Genet. 19:9-12 (2003), Shuey et al., DrugDiscov. Today 7:1040-46 (2002), McManus et al., Nat. Rev. Genet.3:737-747 (2002), Xia et al., Nat. Biotechnol. 20:1006-10 (2002),Plasterk et al., curr. Opin. Genet. Dev. 10:562-7 (2000), Bosher et al.,Nat. Cell Biol. 2:E31-6 (2000), and Hunter, Curr. Biol. 9:R440-442(1999).

A genetic defect leading to increased predisposition or risk fordevelopment of a disease, including cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke, or a defect causing thedisease, may be corrected permanently by administering to a subjectcarrying the defect a nucleic acid fragment that incorporates a repairsequence that supplies the normal/wild-type nucleotide(s) at the site ofthe genetic defect. Such site-specific repair sequence may concompass anRNA/DNA oligonucleotide that operates to promote endogenous repair of asubject's genomic DNA. The administration of the repair sequence may beperformed by an appropriate vehicle, such as a complex withpolyethelenimine, encapsulated in anionic liposomes, a viral vector suchas an adenovirus vector, or other pharmaceutical compositions suitablefor promoting intracellular uptake of the administered nucleic acid. Thegenetic defect may then be overcome, since the chimeric oligonucleotidesinduce the incorporation of the normal sequence into the genome of thesubject, leading to expression of the normal/wild-type gene product. Thereplacement is propagated, thus rendering a permanent repair andalleviation of the symptoms associated with the disease or condition.

The present invention provides methods for identifying compounds oragents that can be used to treat cardiac arrhythmia, e.g. atrialfibrillation and atrial flutter, and stroke. Thus, the variants of theinvention are useful as targets for the identification and/ordevelopment of therapeutic agents. Such methods may include assaying theability of an agent or compound to modulate the activity and/orexpression of a nucleic acid that includes at least one of the variants(markers and/or haplotypes) of the present invention, or the encodedproduct of the nucleic acid. This in turn can be used to identify agentsor compounds that inhibit or alter the undesired activity or expressionof the encoded nucleic acid product. Assays for performing suchexperiments can be performed in cell-based systems or in cell-freesystems, as known to the skilled person. Cell-based systems includecells naturally expressing the nucleic acid molecules of interest, orrecombinant cells that have been genetically modified so as to express acertain desired nucleic acid molecule.

Variant gene expression in a patient can be assessed by expression of avariant-containing nucleic acid sequence (for example, a gene containingat least one variant of the present invention, which can be transcribedinto RNA containing the at least one variant, and in turn translatedinto protein), or by altered expression of a normal/wild-type nucleicacid sequence due to variants affecting the level or pattern ofexpression of the normal transcripts, for example variants in theregulatory or control region of the gene. Assays for gene expressioninclude direct nucleic acid assays (mRNA), assays for expressed proteinlevels, or assays of collateral compounds involved in a pathway, forexample a signal pathway. Furthermore, the expression of genes that areup- or down-regulated in response to the signal pathway can also beassayed. One embodiment includes operably linking a reporter gene, suchas luciferase, to the regulatory region of the gene(s) of interest.

Modulators of gene expression can in one embodiment be identified when acell is contacted with a candidate compound or agent, and the expressionof mRNA is determined. The expression level of mRNA in the presence ofthe candidate compound or agent is compared to the expression level inthe absence of the compound or agent. Based on this comparison,candidate compounds or agents for treating disorders such as atrialfibrillation, atrial flutter and stroke can be identified as thosemodulating the gene expression of the variant gene. When expression ofmRNA or the encoded protein is statistically significantly greater inthe presence of the candidate compound or agent than in its absence,then the candidate compound or agent is identified as a stimulator orup-regulator of expression of the nucleic acid. When nucleic acidexpression or protein level is statistically significantly less in thepresence of the candidate compound or agent than in its absence, thenthe candidate compound is identified as an inhibitor or down-regulatorof the nucleic acid expression.

The invention further provides methods of treatment using a compoundidentified through drug (compound and/or agent) screening as a genemodulator (i.e. stimulator and/or inhibitor of gene expression).

In a further aspect of the present invention, a pharmaceutical pack(kit) is provided, the pack comprising a therapeutic agent and a set ofinstructions for administration of the therapeutic agent to humansdiagnostically tested for one or more variants of the present invention,as disclosed herein. The therapeutic agent can be a small molecule drug,an antibody, a peptide, an antisense or RNAi molecule, or othertherapeutic molecules. In one embodiment, an individual identified as acarrier of at least one variant of the present invention is instructedto take a prescribed dose of the therapeutic agent. In one suchembodiment, an individual identified as a homozygous carrier of at leastone variant of the present invention is instructed to take a prescribeddose of the therapeutic agent. In another embodiment, an individualidentified as a non-carrier of at least one variant of the presentinvention is instructed to take a prescribed dose of the therapeuticagent.

Methods of Assessing Probability of Response to Therapeutic Agents,Methods of Monitoring Progress of Treatment and Methods of Treatment

As is known in the art, individuals can have differential responses to aparticular therapy (e.g., a therapeutic agent or therapeutic method).Pharmacogenomics addresses the issue of how genetic variations (e.g.,the variants (markers and/or haplotypes) of the present invention)affect drug response, due to altered drug disposition and/or abnormal oraltered action of the drug. Thus, the basis of the differential responsemay be genetically determined in part. Clinical outcomes due to geneticvariations affecting drug response may result in toxicity of the drug incertain individuals (e.g., carriers or non-carriers of the geneticvariants of the present invention), or therapeutic failure of the drug.Therefore, the variants of the present invention may determine themanner in which a therapeutic agent and/or method acts on the body, orthe way in which the body metabolizes the therapeutic agent.

Accordingly, in one embodiment, the presence of a particular allele at apolymorphic site or haplotype is indicative of a different, e.g. adifferent response rate, to a particular treatment modality. This meansthat a patient diagnosed with cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke, and carrying a certainallele at a polymorphic or haplotype of the present invention (e.g., theat-risk and protective alleles and/or haplotypes of the invention) wouldrespond better to, or worse to, a specific therapeutic, drug and/orother therapy used to treat the disease. Therefore, the presence orabsence of the marker allele or haplotype could aid in deciding whattreatment should be used for a the patient. For example, for a newlydiagnosed patient, the presence of a marker or haplotype of the presentinvention may be assessed (e.g., through testing DNA derived from ablood sample, as described herein). If the patient is positive for amarker allele or haplotype at (that is, at least one specific allele ofthe marker, or haplotype, is present), then the physician recommends oneparticular therapy, while if the patient is negative for the at leastone allele of a marker, or a haplotype, then a different course oftherapy may be recommended (which may include recommending that noimmediate therapy, other than serial monitoring for progression of thedisease, be performed). Thus, the patient's carrier status could be usedto help determine whether a particular treatment modality should beadministered. The value lies within the possibilities of being able todiagnose the disease at an early stage, to select the most appropriatetreatment, and provide information to the clinician aboutprognosis/aggressiveness of the disease in order to be able to apply themost appropriate treatment.

Treatment of Atrial Fibrillation and Atrial Flutter is GenerallyDirected by Two Main Objectives: (i) to Prevent Stroke and (ii) to TreatSymptoms. (i) Stroke Prevention

Anticoagulation is the therapy of choice for stroke prevention in atrialfibrillation and is indicated for the majority of patients with thisarrhythmia. The only patients for whom anticoagulation is not stronglyrecommended are those younger than 65 years old who are consideredlow-risk, i.e., they have no organic heart disease, no hypertension, noprevious history of stroke or transient ischemic attacks and nodiabetes. This group as a whole has a lower risk of stroke and strokeprevention with aspirin is generally recommended. For all otherpatients, anticoagulation is indicated whether the atrial fibrillationis permanent, recurrent paroxysmal or recurrent persistent. It cannot begeneralized how patients who present with their first episode ofparoxysmal atrial fibrillation should be treated and the decision needsto be individualized for each patient. Anticoagulation is also indicatedeven when the patient with atrial fibrillation is felt to be maintainedin sinus rhythm with antiarrhythmic therapy (rhythm controlled) sincethis type of therapy does not affect stroke risk.

Anticoagulants.

Anticoagulation is recommended in atrial fibrillation, as detailedabove, for prevention of cardioembolism and stroke. The most widelystudied oral anticoagulant is warfarin and this medication isuniversally recommended for chronic oral anticoagulation in atrialfibrillation. Warfarin has few side effects aside from the risk ofbleeding but requires regular and careful monitoring of blood valuesduring therapy (to measure the effect of the anticoagulation). The oralanticoagulant ximelagatran showed promise in stroke prevention inpatients with atrial fibrillation and had the advantage of not requiringregular monitoring like warfarin. Ximelagatran was found however tocause unexplained liver injury and was withdrawn from the market in2006. Several agents are available for intravenous and/or subcutaneoustherapy, including heparin and the low molecular weight heparins (e.g.enoxaparin, dalteparin, tinzaparin, ardeparin, nadroparin andreviparin). These medications are recommended when rapid initiation ofanticoagulation is necessary or if oral anticoagulation therapy has tobe interrupted in high risk patients or for longer than one week inother patients for example due to a series of procedures. Otherparenteral anticoagulants are available but not specifically recommendedas therapy in atrial fibrillation; e.g., the factor Xa inhibitorsfondaparinux and idraparinux, the thrombin-inhibitors lepirudin,bivalirudin and argatroban as well as danaparoid.

(ii) Symptom Control.

Medical and surgical therapy applied to control symptoms of atrialfibrillation is tailored to the individual patient and consists of heartrate and/or rhythm control with medications, radiofrequency ablationand/or surgery.

Antiarrhythmic Medications.

In general terms, antiarrhythmic agents are used to suppress abnormalrhythms of the heart that are characteristic of cardiac arrhythmias,including atrial fibrillation and atrial flutter. One classification ofantiarrhythmic agents is the Vaughan Williams classification, in whichfive main categories of antiarrhythmic agents are defined. Class Iagents are fast sodium channel blockers and are subclassified based onkinetics and strength of blockade as well as their effect onrepolarization. Class Ia includes disopyramide, moricizine, procainamideand quinidine. Class Ib agents are lidocaine, mexiletine, tocainide, andphenytoin. Class Ic agents are encainide, flecainide, propafenone,ajmaline, cibenzoline and detajmium. Class II agents are beta blockers,they block the effects of catecholamines at beta-adrenergic receptors.Examples of beta blockers are esmolol, propranolol, metoprolol,alprenolol, atenolol, carvedilol, bisoprolol, acebutolol, nadolol,pindolol, labetalol, oxprenotol, penbutolol, timolol, betaxolol,cartelol, sotalol and levobunolol. Class III agents have mixedproperties but are collectively potassium channel blockers and prolongrepolarization. Medications in this category are amiodarone, azimilide,bretylium, dofetilide, tedisamil, ibutilide, sematilide, sotalol,N-acetyl procainamide, nifekalant hydrochloride, vernakalant andambasilide. Class IV agents are calcium channel blockers and includeverapamil, mibefradil and diltiazem. Finally, class V consists ofmiscellaneous antiarrhythmics and includes digoxin and adenosine.

Heart Rate Control

Pharmacologic measures for maintenance of heart rate control includebeta blockers, calcium channel blockers and digoxin. All thesemedications slow the electrical conduction through the atrioventricularnode and slow the ventricular rate response to the rapid atrialfibrillation. Some antiarrhythmics used primarily for rhythm control(see below) also slow the atrioventricular node conduction rate and thusthe ventricular heart rate response. These include some class III and Icmedications such as amiodarone, sotalol and flecainide.

Cardioversion.

Cardioversion of the heart rhythm from atrial fibrillation or atrialflutter to sinus rhythm can be achieved electrically, with synchronizeddirect-current cardioversion, or with medications such as ibutilide,amiodarone, procainamide, propafenone and flecainide.

Heart Rhythm Control

Medications used for maintenance of sinus rhythm, i.e. rhythm control,include mainly antiarrhythmic medications from classes III, Ia and Ic.Examples are sotalol, amiodarone and dofetilide from class III,disopyramide, procainamide and quinidine from class Ia and flecinide andpropafenone from class Ic. Treatment with these antiarrhythmicmedications is complicated, can be hazardous, and should be directed byphysicians specifically trained to use these medications. Many of theantiarrhythmics have serious side effects and should only be used inspecific populations. For example, class Ic medications should not beused in patients with coronary artery disease and even if they cansuppress atrial fibrillation, they can actually promote rapidventricular response in atrial flutter. Class Ia medications can be usedas last resort in patients without structural heart diseases. Sotalol(as most class III antiarrhythmics) can cause significant prolongationof the QT interval, specifically in patients with renal failure, andpromote serious ventricular arrhythmias. Both sotalol and dofetilide aswell as the Ia medications need to be initiated on an inpatient basis tomonitor the QT interval. Although amiodarone is usually well toleratedand is widely used, amiodarone has many serious side effects withlong-term therapy.

How Genetic Testing May Directly Affect Choice of Treatment

When individuals present with their first (diagnosed) episode ofparoxysmal atrial fibrillation and either spontaneously convert to sinusrhythm or undergo electrical or chemical cardioversion less than 48hours into the episode, the decision to initiate, or not to initiate,anticoagulation therapy, is individualized based on the risk profile ofthe patient in question and the managing physicians preference. This canbe a difficult choice to make since committing the patient toanticoagulation therapy has a major impact on the patients life. Oftenthe choice is made to withhold anticoagulation in such a situation andthis may be of no significant consequence to the patient. On the otherhand the patient may later develop a stroke and the opportunity ofprevention may thus have been missed. In such circumstances, knowingthat the patient is a carrier of the at-risk variant may be of greatsignificance and support initiation of anticoagulation treatment.

Individuals who are diagnosed with atrial fibrillation under the age of65 and are otherwise considered low risk for stroke, i.e. have noorganic heart disease, no hypertension, no diabetes and no previoushistory of stroke, are generally treated with aspirin only forstroke-prevention and not anticoagulation. If such a patient is found tobe carrier for the at-risk variants described herein, this could beconsidered support for initiating anticoagulation earlier than otherwiserecommended. This would be a reasonable consideration since the resultsof stroke from atrial fibrillation can be devastating.

Ischemic stroke is generally classified into five subtypes based onsuspected cause; large artery atherosclerosis, small artery occlusion,cardioembolism (majority due to atrial fibrillation), stroke of otherdetermined cause and stroke of undetermined cause (either no cause foundor more than 1 plausible cause). Importantly, strokes due tocardioembolism have the highest recurrence, are most disabling and areassociated with the lowest survival. It is therefore imperative not tooverlook atrial fibrillation as the major cause of stroke, particularlysince treatment measures vary based on the subtype. Therefore, if anindividual is diagnosed with stroke or a transient ischemic attack and aplausible cause is not identified despite standard work-up, knowing thatthe patient is a carrier of the at-risk variant may be of great valueand support either initiation of anticoagulation treatment or moreaggressive diagnostic testing in the attempt to diagnose atrialfibrillation.

Furthermore, the markers of the present invention can be used toincrease power and effectiveness of clinical trials. Thus, individualswho are carriers of at least one at-risk variant of the presentinvention, i.e. individuals who are carriers of at least one allele ofat least one polymorphic marker conferring increased risk of developingcardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/orstroke may be more likely to respond to a particular treatment modality,e.g., as described in the above. In one embodiment, individuals whocarry at-risk variants for gene(s) in a pathway and/or metabolic networkfor which a particular treatment (e.g., small molecule drug) istargeting, are more likely to be responders to the treatment. In anotherembodiment, individuals who carry at-risk variants for a gene, whichexpression and/or function is altered by the at-risk variant, are morelikely to be responders to a treatment modality targeting that gene, itsexpression or its gene product. This application can improve the safetyof clinical trials, but can also enhance the chance that a clinicaltrial will demonstrate statistically significant efficacy, which may belimited to a certain sub-group of the population. Thus, one possibleoutcome of such a trial is that carriers of certain genetic variants,e.g., the markers and haplotypes of the present invention, arestatistically significantly likely to show positive response to thetherapeutic agent, i.e. experience alleviation of symptoms associatedwith cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter)and/or stroke when taking the therapeutic agent or drug as prescribed.

In a further aspect, the markers and haplotypes of the present inventioncan be used for targeting the selection of pharmaceutical agents forspecific individuals. Personalized selection of treatment modalities,lifestyle changes or combination of the two, can be realized by theutilization of the at-risk variants of the present invention. Thus, theknowledge of an individual's status for particular markers of thepresent invention, can be useful for selection of treatment options thattarget genes or gene products affected by the at-risk variants of theinvention. Certain combinations of variants may be suitable for oneselection of treatment options, while other gene variant combinationsmay target other treatment options. Such combination of variant mayinclude one variant, two variants, three variants, or four or morevariants, as needed to determine with clinically reliable accuracy theselection of treatment module.

Computer-Implemented Applications

The present invention also relates to computer-implemented applicationsof the polymorphic markers and haplotypes described herein to beassociated with cardiac arrhythmia (e.g., atrial fibrillation and atrialflutter) and stroke. Such applications can be useful for storing,manipulating or otherwise analyzing genotype data that is useful in themethods of the invention. One example pertains to storing genotypeinformation derived from an individual on readable media, so as to beable to provide the genotype information to a third party (e.g., theindividual), or for deriving information from the genotype data, e.g.,by comparing the genotype data to information about genetic risk factorscontributing to increased susceptibility to cardiac arrhythmia (e.g.,atrial fibrillation and atrial flutter) and stroke, and reportingresults based on such comparison.

One such aspect relates to computer-readable media. In general terms,such medium has capabilities of storing (i) identifier information forat least one polymorphic marker or a haplotye; (ii) an indicator of thefrequency of at least one allele of said at least one marker, or thefrequency of a haplotype, in individuals with cardiac arrhythmia (e.g.,atrial fibrillation and atrial flutter) and/or stroke; and an indicatorof the frequency of at least one allele of said at least one marker, orthe frequency of a haplotype, in a reference population. The referencepopulation can be a disease-free population of individuals.Alternatively, the reference population is a random sample from thegeneral population, and is thus representative of the population atlarge. The frequency indicator may be a calculated frequency, a count ofalleles and/or haplotype copies, or normalized or otherwise manipulatedvalues of the actual frequencies that are suitable for the particularmedium.

Additional information about the individual can be stored on the medium,such as ancestry information, information about sex, physical attributesor characteristics (including height and weight), biochemicalmeasurements (such as blood pressure, blood lipid levels, fastingglucose levels, insulin response measurements), biomarker results, orother useful information that is desirable to store or manipulate in thecontext of the genotype status of a particular individual.

The invention furthermore relates to an apparatus that is suitable fordetermination or manipulation of genetic data useful for determining asusceptibility to cardiac arrhythmia (e.g., atrial fibrillation andatrial flutter) and stroke in a human individual. Such an apparatus caninclude a computer-readable memory, a routine for manipulating datastored on the computer-readable memory, and a routine for generating anoutput that includes a measure of the genetic data. Such measure caninclude values such as allelic or haplotype frequencies, genotypecounts, sex, age, phenotype information, values for odds ratio (OR) orrelative risk (RR), population attributable risk (PAR), or other usefulinformation that is either a direct statistic of the original genotypedata or based on calculations based on the genetic data.

The above-described applications can all be practiced with the markersand haplotypes of the invention that have in more detail been describedwith respect to methods of assessing susceptibility to cardiacarrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke.Thus, these applications can in general be reduced to practice usingmarkers listed in Tables 5, Table 4, Table 9, and Table 19, and markersin linkage disequilibrium therewith. In one embodiment, the markers orhaplotypes are present within the genomic segment whose sequences is setforth in SEQ ID NO:50. In another embodiment, the markers or haplotypescomprise at least one marker selected from the markers set forth inTable 19. In another embodiment, the markers and haplotypes comprise atleast one marker selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ IDNO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464(SEQ ID NO:41), and rs13143308 (SEQ ID NO:51), optionally includingmarkers in linkage disequilibrium therewith. In one such embodiment,linkage disequilibrium is defined by numerical values for r² of greaterthan 0.1. In another such embodiment, linkage disequilibrium is definedby numerical values for r² of greater than 0.2. In another embodiment,the marker or haplotype comprises at least one allele selected fromalleles-2, -4 and/or -8 in marker D4S406, allele A of marker rs2634073,allele T of marker rs2200733, allele T of marker rs2220427, allele T ofmarker rs10033464, and/or allele G of marker rs13143308

Nucleic Acids and Polypeptides

The nucleic acids and polypeptides described herein can be used inmethods an kits of the present invention, as described in the above.

An “isolated” nucleic acid molecule, as used herein, is one that isseparated from nucleic acids that normally flank the gene or nucleotidesequence (as in genomic sequences) and/or has been completely orpartially purified from other transcribed sequences (e.g., as in an RNAlibrary). For example, an isolated nucleic acid of the invention can besubstantially isolated with respect to the complex cellular milieu inwhich it naturally occurs, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized. In some instances, the isolated material willform part of a composition (for example, a crude extract containingother substances), buffer system or reagent mix. In other circumstances,the material can be purified to essential homogeneity, for example asdetermined by polyacrylamide gel electrophoresis (PAGE) or columnchromatography (e.g., HPLC). An isolated nucleic acid molecule of theinvention can comprise at least about 50%, at least about 80% or atleast about 90% (on a molar basis) of all macromolecular speciespresent. With regard to genomic DNA, the term “isolated” also can referto nucleic acid molecules that are separated from the chromosome withwhich the genomic DNA is naturally associated. For example, the isolatednucleic acid molecule can contain less than about 250 kb, 200 kb, 150kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid moleculein the genomic DNA of the cell from which the nucleic acid molecule isderived.

The nucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated. Thus, recombinant DNAcontained in a vector is included in the definition of “isolated” asused herein. Also, isolated nucleic acid molecules include recombinantDNA molecules in heterologous host cells or heterologous organisms, aswell as partially or substantially purified DNA molecules in solution.“Isolated” nucleic acid molecules also encompass in vivo and in vitroRNA transcripts of the DNA molecules of the present invention. Anisolated nucleic acid molecule or nucleotide sequence can include anucleic acid molecule or nucleotide sequence that is synthesizedchemically or by recombinant means. Such isolated nucleotide sequencesare useful, for example, in the manufacture of the encoded polypeptide,as probes for isolating homologous sequences (e.g., from other mammalianspecies), for gene mapping (e.g., by in situ hybridization withchromosomes), or for detecting expression of the gene in tissue (e.g.,human tissue), such as by Northern blot analysis or other hybridizationtechniques.

The invention also pertains to nucleic acid molecules that hybridizeunder high stringency hybridization conditions, such as for selectivehybridization, to a nucleotide sequence described herein (e.g., nucleicacid molecules that specifically hybridize to a nucleotide sequencecontaining a polymorphic site associated with a marker or haplotypedescribed herein). In one embodiment, the invention includes variantsthat hybridize under high stringency hybridization and wash conditions(e.g., for selective hybridization) to a nucleotide sequence thatcomprises the nucleotide sequence of LD Block C04 (SEQ ID NO:50). Suchnucleic acid molecules can be detected and/or isolated by allele- orsequence-specific hybridization (e.g., under high stringencyconditions). Stringency conditions and methods for nucleic acidhybridizations are well known to the skilled person (see, e.g., CurrentProtocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons,(1998), and Kraus, M. and Aaronson, S., Methods Enzymol., 200:546-556(1991), the entire teachings of which are incorporated by referenceherein.

The percent identity of two nucleotide or amino acid sequences can bedetermined by aligning the sequences for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first sequence). Thenucleotides or amino acids at corresponding positions are then compared,and the percent Identity between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %identity=# of identical positions/total # of positions×100). In certainembodiments, the length of a sequence aligned for comparison purposes isat least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%, of the length of the referencesequence. The actual comparison of the two sequences can be accomplishedby well-known methods, for example, using a mathematical algorithm. Anon-limiting example of such a mathematical algorithm is described inKarlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993). Such an algorithm is incorporated into the NBLAST and XBLASTprograms (version 2.0), as described in Altschul, S. et al., NucleicAcids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLAST) can be used. See the website on the world wide web atncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparisoncan be set at score=100, wordlength=12, or can be varied (e.g., W=5 orW=20).

Other examples include the algorithm of Myers and Miller, CABIOS (1989),ADVANCE and ADAM as described in Torellis, A. and Robotti, C., Comput.Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson, W. andLipman, D., Proc. Natl. Acad. Sci. USA, 85:2444-48 (1988). In anotherembodiment, the percent identity between two amino acid sequences can beaccomplished using the GAP program in the GCG software package(Accelrys, Cambridge, UK).

The present invention also provides isolated nucleic acid molecules thatcontain a fragment or portion that hybridizes under highly stringentconditions to a nucleic acid that comprises, or consists of, thenucleotide sequence of LD Block C04 (SEQ ID NO:50), or a nucleotidesequence comprising, or consisting of, the complement of the nucleotidesequence of LD Block C04 (SEQ ID NO:50), wherein the nucleotide sequencecomprises at least one polymorphic allele contained in the markers andhaplotypes described herein. The nucleic acid fragments of the inventionare at least about 15, at least about 18, 20, 23 or 25 nucleotides, andcan be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides inlength.

The nucleic acid fragments of the invention are used as probes orprimers in assays such as those described herein. “Probes” or “primers”are oligonucleotides that hybridize in a base-specific manner to acomplementary strand of a nucleic acid molecule. In addition to DNA andRNA, such probes and primers include polypeptide nucleic acids (PNA), asdescribed in Nielsen, P. et al., Science 254:1497-1500 (1991). A probeor primer comprises a region of nucleotide sequence that hybridizes toat least about 15, typically about 20-25, and in certain embodimentsabout 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule.In one embodiment, the probe or primer comprises at least one allele ofat least one polymorphic marker or at least one haplotype describedherein, or the complement thereof. In particular embodiments, a probe orprimer can comprise 100 or fewer nucleotides; for example, in certainembodiments from 6 to 50 nucleotides, or, for example, from 12 to 30nucleotides. In other embodiments, the probe or primer is at least 70%identical, at least 80% identical, at least 85% identical, at least 90%identical, or at least 95% identical, to the contiguous nucleotidesequence or to the complement of the contiguous nucleotide sequence. Inanother embodiment, the probe or primer is capable of selectivelyhybridizing to the contiguous nucleotide sequence or to the complementof the contiguous nucleotide sequence. Often, the probe or primerfurther comprises a label, e.g., a radioisotope, a fluorescent label, anenzyme label, an enzyme co-factor label, a magnetic label, a spin label,an epitope label.

The nucleic acid molecules of the invention, such as those describedabove, can be identified and isolated using standard molecular biologytechniques well known to the skilled person. The amplified DNA can belabeled (e.g., radiolabeled) and used as a probe for screening a cDNAlibrary derived from human cells. The cDNA can be derived from mRNA andcontained in a suitable vector. Corresponding clones can be isolated,DNA can obtained following in vivo excision, and the cloned insert canbe sequenced in either or both orientations by art-recognized methods toidentify the correct reading frame encoding a polypeptide of theappropriate molecular weight. Using these or similar methods, thepolypeptide and the DNA encoding the polypeptide can be isolated,sequenced and further characterized.

In general, the isolated nucleic acid sequences of the invention can beused as molecular weight markers on Southern gels, and as chromosomemarkers that are labeled to map related gene positions. The nucleic acidsequences can also be used to compare with endogenous DNA sequences inpatients to identify cardiac arrhythmia (e.g., atrial fibrillation oratrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia(e.g., atrial fibrillation or atrial flutter) and/or stroke, and asprobes, such as to hybridize and discover related DNA sequences or tosubtract out known sequences from a sample (e.g., subtractivehybridization). The nucleic acid sequences can further be used to deriveprimers for genetic fingerprinting, to raise anti-polypeptide antibodiesusing immunization techniques, and/or as an antigen to raise anti-DNAantibodies or elicit immune responses.

Antibodies

Polyclonal antibodies and/or monoclonal antibodies that specificallybind one form of the gene product but not to the other form of the geneproduct are also provided. Antibodies are also provided which bind aportion of either the variant or the reference gene product thatcontains the polymorphic site or sites. The term “antibody” as usedherein refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that containantigen-binding sites that specifically bind an antigen. A molecule thatspecifically binds to a polypeptide of the invention is a molecule thatbinds to that polypeptide or a fragment thereof, but does notsubstantially bind other molecules in a sample, e.g., a biologicalsample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind to a polypeptide of theinvention. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of a polypeptide ofthe invention. A monoclonal antibody composition thus typically displaysa single binding affinity for a particular polypeptide of the inventionwith which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a desired immunogen, e.g., polypeptide of theinvention or a fragment thereof. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules directed against thepolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein, Nature256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al.,Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp.77-96) or trioma techniques. The technology for producing hybridomas iswell known (see generally Current Protocols in Immunology (1994) Coliganet al., (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, animmortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds a polypeptide of the invention.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the invention (see, e.g.,Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052(1977); R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); andLerner, Yale J. Biol. Med. 54:387-402 (1981)). Moreover, the ordinarilyskilled worker will appreciate that there are many variations of suchmethods that also would be useful.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody to a polypeptide of the invention can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with the polypeptideto thereby isolate immunoglobulin library members that bind thepolypeptide. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO90/02809; Fuchs et al., Bio/Technology 9: 1370-1372 (1991); Hay et al.,Hum. Antibod. Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, antibodies of the invention (e.g., a monoclonal antibody)can be used to isolate a polypeptide of the invention by standardtechniques, such as affinity chromatography or immunoprecipitation. Apolypeptide-specific antibody can facilitate the purification of naturalpolypeptide from cells and of recombinantly produced polypeptideexpressed in host cells. Moreover, an antibody specific for apolypeptide of the invention can be used to detect the polypeptide(e.g., in a cellular lysate, cell supernatant, or tissue sample) inorder to evaluate the abundance and pattern of expression of thepolypeptide. Antibodies can be used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Theantibody can be coupled to a detectable substance to facilitate itsdetection. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibodies may also be useful in pharmacogenomic analysis. In suchembodiments, antibodies against variant proteins encoded by nucleicacids according to the invention, such as variant proteins that areencoded by nucleic acids that contain at least one polymorphic marker ofthe invention, can be used to identify individuals that require modifiedtreatment modalities.

Antibodies can furthermore be useful for assessing expression of variantproteins in disease states, such as in active stages of a disease, or inan individual with a predisposition to a disease related to the functionof the protein, in particular cardiac arrhythmia (e.g., atrialfibrillation or atrial flutter) and/or stroke. Antibodies specific for avariant protein of the present invention that is encoded by a nucleicacid that comprises at least one polymorphic marker or haplotype asdescribed herein can be used to screen for the presence of the variantprotein, for example to screen for a predisposition to cardiacarrhythmia (e.g., atrial fibrillation or atrial flutter) and/or strokeas indicated by the presence of the variant protein.

Antibodies can be used in other methods. Thus, antibodies are useful asdiagnostic tools for evaluating proteins, such as variant proteins ofthe invention, in conjunction with analysis by electrophoretic mobility,isoelectric point, tryptic or other protease digest, or for use in otherphysical assays known to those skilled in the art. Antibodies may alsobe used in tissue typing. In one such embodiment, a specific variantprotein has been correlated with expression in a specific tissue type,and antibodies specific for the variant protein can then be used toidentify the specific tissue type.

Subcellular localization of proteins, including variant proteins, canalso be determined using antibodies, and can be applied to assessaberrant subcellular localization of the protein in cells in varioustissues. Such use can be applied in genetic testing, but also inmonitoring a particular treatment modality. In the case where treatmentis aimed at correcting the expression level or presence of the variantprotein or aberrrant tissue distribution or developmental expression ofthe variant protein, antibodies specific for the variant protein orfragments thereof can be used to monitor therapeutic efficacy.

Antibodies are further useful for inhibiting variant protein function,for example by blocking the binding of a variant protein to a bindingmolecule or partner. Such uses can also be applied in a therapeuticcontext in which treatment involves inhibiting a variant protein'sfunction. An antibody can be for example be used to block orcompetitively inhibit binding, thereby modulating (i.e., agonizing orantagonizing) the activity of the protein. Antibodies can be preparedagainst specific protein fragments containing sites required forspecific function or against an intact protein that is associated with acell or cell membrane. For administration in vivo, an antibody may belinked with an additional therapeutic payload, such as radionuclide, anenzyme, an immunogenic epitope, or a cytotoxic agent, includingbacterial toxins (diphteria or plant toxins, such as ricin). The in vivohalf-life of an antibody or a fragment thereof may be increased bypegylation through conjugation to polyethylene glycol.

The present invention further relates to kits for using antibodies inthe methods described herein. This includes, but is not limited to, kitsfor detecting the presence of a variant protein in a test sample. Onepreferred embodiment comprises antibodies such as a labelled orlabelable antibody and a compound or agent for detecting variantproteins in a biological sample, means for determining the amount or thepresence and/or absence of variant protein in the sample, and means forcomparing the amount of variant protein in the sample with a standard,as well as instructions for use of the kit.

The present invention will now be exemplified by the followingnon-limiting example.

EXEMPLIFICATION Example 1 Identification of At-Risk Variants for AtrialFibrillation on Chromosome 4

The following contains description of the identification ofsusceptibility factors found to be associated with atrial fibrillationand stroke through single-point analysis of SNP markers.

Methods.

The study was approved by the Data Protection Commission of Iceland andthe National Bioethics Committee.

Icelandic AF Cohort.

The patients were all diagnosed with AF at the Landspitali UniversityHospital in Reykjavik, Iceland, from 1987 to 2003. Diagnoses wereconfirmed by a 12 lead electrocardiogram demonstrating no P waves andirregularly irregular R-R intervals. All ECGs were manually read by acardiologist.

Icelandic Stroke Cohort.

The stroke cohort was derived from two major hospitals in Iceland andthe Icelandic Heart Association. Patients with hemorrhagic strokerepresented 6% of all patients (patients with the Icelandic type ofhereditary cerebral hemorrhage with amyloidosis and patients withsubarachnoid hemorrhage were excluded). Ischemic stroke accounted for67% of the total patients and TIAs 27%. The distribution of strokesubtypes in this study is similar to that reported in other Caucasianpopulations (Mohr, J. P., et al., Neurology, 28:754-762 (1978); L. R.Caplan, In Stroke, A Clinical Approach (Butterworth-Heinemann, Stoneham,Mass., ed 3, (1993)).

Genotyping.

A genome-wide scan of 437 Icelandic individuals diagnosed with AtrialFibrillation (AF) and 7406 population controls was performed usingInfinium HumanHap300 SNP chips from Illumina for assaying approximately317,000 single nucleotide polymorphisms (SNPs) on a single chip(Illumina, San Diego, Calif., USA), SNP genotyping for replication inother case-control cohorts was carried using the Centaurus platform(Nanogen). A total of 347 individuals diagnosed with Stroke and 7497controls was also performed for SNPs within the LD Block found to beassociated with Atrial Fibrillation.

Statistical Methods for Association Analysis.

For single marker association to atrial fibrillation or stroke, we useda likelihood ratio test to calculate a two-sided p-value for eachallele. We calculated relative risk (RR) and population attributablerisk (PAR) assuming a multiplicative model (C. T. Falk, P. Rubinstein,Ann Hum Genet 51 (Pt 3), 227 (1987); J. D. Terwilliger, J. Ott, HumHered 42, 337 (1992)). For the CEPH Caucasian HapMap data, we calculatedLD between pairs of SNPs using the standard definition of D′ (R. C.Lewontin, Genetics 50, 757 (1964)) and R² W. G. Hill, A. Robertson,Genetics 60, 615 (November, 1968). When plotting all SNP combinations toelucidate the LD structure in a particular region, we plotted D′ in theupper left corner and p-values in the lower right corner. In the LDplots we present, the markers are plotted equidistantly rather thanaccording to their physical positions.

Results Genome-Wide Association Study

We successfully genotyped 437 Icelandic Atrial Fibriallation patientsand 7406 population control individuals using the Illumina 330K chip.Association analysis was performed for single SNPs. The most significantassociation was found for markers rs2220427 and rs2220733, both of whichgive p-values close to 10⁻⁹. The value for rs2220427 is significantafter correcting for the number of tests performed, i.e. the associationis significant at the genome-wide level.

There is an apparent excess of homozygotes in affected individuals. Wereject both the multiplicative model (P=0.002) and the recessive model(P=0.001). The best fitting model gives risk 1.46 to heterozygouscarriers and 5.17 to homozygous carriers. The (uncorrected) P-valuecomparing this full model to the null model of no association is5.43e-11. These data show that individuals with two copies of theat-risk allele are at greater risk than expected based on a simplemultiplicative model.

Fitting an age at onset model for all genotypes gives a P-value of4.84e-5. Heterozygotes are estimated to have onset 1.4090 years earlierthan non-carriers and homozygote carriers are estimated to have onset9.6126 years earlier than non-carriers. This shows a significant effectof age at onset—individuals carrying the at-risk variant are atsignificant risk of developing AF at a younger age than individuals whoare non-carriers of the at-risk allele.

Investigating markers in the vicinity of rs2220427, we realized that themicrosatellite marker D4S406 can be used as a surrogate marker forrs2220427. In particular, alleles-2, -4 and -8 (with respect to the CEPHreference) were found to be sufficient to tag the SNP based on haplotypefrequencies:

TABLE 1 Relationship between rs2220427 and D4S406 Haplotype Frequency MSallele SNP Allele 7.55E−05 −2 D4S406 2 rs2220427 0.000109727 16 D4S406 4rs2220427 0.000148065 −6 D4S406 4 rs2220427 0.000149685 20 D4S406 2rs2220427 0.000149756 −4 D4S406 2 rs2220427 0.000210154 8 D4S406 4rs2220427 0.000225802 −8 D4S406 2 rs2220427 0.000227036 4 D4S406 4rs2220427 0.000299371 18 D4S406 2 rs2220427 0.000899281 0 D4S406 4rs2220427 0.00203518 −4 D4S406 4 rs2220427 0.00673851 −6 D4S406 2rs2220427 0.0245484 2 D4S406 2 rs2220427 0.0394983 −2 D4S406 4 rs22204270.0422112 14 D4S406 2 rs2220427 0.0594303 0 D4S406 2 rs2220427 0.0762831−8 D4S406 4 rs2220427 0.0855451 6 D4S406 2 rs2220427 0.0949753 12 D4S4062 rs2220427 0.100105 16 D4S406 2 rs2220427 0.145942 4 D4S406 2 rs22204270.155838 8 D4S406 2 rs2220427 0.164354 10 D4S406 2 rs2220427Thus, for individuals typed for the D4S406 marker but not rs222047,merging the -2, -4 and -8 alleles leads to a very good estimate of thefrequency of the 4 allele of the SNP.

We analyzed an Icelandic replication cohort for AF, comprised of 1269cases and 69,070 controls, in this fashion. The results are quitedramatic in that the association is accompanied by a p-value of 2.94e-14and a relative risk (multiplicative model) of 1.53. Thus, our initialfinding has been replicated in an independent Icelandic cohort.

TABLE 2 Association of AF patients to Chromosome 4 (LD Block C04). Shownare values for RR under the multiplicative model. Relative p-value RiskAff freq Con freq Allele Marker 0.16839039 0.8473 0.902746 0.916352 3rs10033464 0.16839039 1.1802 0.097254 0.083648 4 rs10033464 0.242753460.8746 0.098398 0.110939 1 rs13105878 0.24275346 1.1433 0.9016020.889061 2 rs13105878 4.89E−06 1.3816 0.441648 0.364078 1 rs131411904.89E−06 0.7238 0.558352 0.635922 3 rs13141190 1.25E−06 0.6905 0.6773460.752498 1 rs1448817 1.25E−06 1.4483 0.322654 0.247502 3 rs14488170.00995996 0.7903 0.811213 0.844653 2 rs16997168 0.00995996 1.26540.188787 0.155347 4 rs16997168 1.07E−09 0.5601 0.811213 0.884688 2rs2200733 1.07E−09 1.7855 0.188787 0.115312 4 rs2200733 7.78E−10 0.5570.810345 0.884673 2 rs2220427 7.78E−10 1.7953 0.189655 0.115327 4rs2220427 9.75E−08 1.5803 0.236239 0.163692 1 rs2634073 9.75E−08 0.63280.763761 0.836308 3 rs2634073 0.96927011 0.9968 0.768879 0.769444 1rs2723296 0.96927011 1.0032 0.231121 0.230556 3 rs2723296 0.032817130.8529 0.665904 0.700311 2 rs2723316 0.03281713 1.1724 0.334096 0.2996894 rs2723316 0.01803327 1.1855 0.635632 0.595393 1 rs3853444 0.018033270.8435 0.364368 0.404607 3 rs3853444 0.40105752 0.9214 0.146789 0.157342 rs4576077 0.40105752 1.0853 0.853211 0.84266 4 rs4576077 0.932696211.0084 0.145309 0.144275 1 rs6419178 0.93269621 0.9917 0.854691 0.8557253 rs6419178

TABLE 3 Association of Stroke to markers within LD Block C04 (SEQ ID NO:50) Relative p-value Risk Aff freq Con freq Allele Marker 0.377018520.8872 0.90634 0.916022 3 rs10033464 0.37701852 1.1272 0.09366 0.0839784 rs10033464 0.2838194 0.8717 0.097983 0.110807 1 rs13105878 0.28381941.1472 0.902017 0.889193 2 rs13105878 0.01534596 1.2123 0.4121040.366378 1 rs13141190 0.01534596 0.8249 0.587896 0.633622 3 rs131411900.04856224 0.8418 0.716138 0.7498 1 rs1448817 0.04856224 1.1879 0.2838620.2502 3 rs1448817 0.14450185 0.8599 0.822767 0.843717 2 rs169971680.14450185 1.1629 0.177233 0.156283 4 rs16997168 0.00374992 0.7240.84438 0.882271 2 rs2200733 0.00374992 1.3812 0.15562 0.117729 4rs2200733 0.0025713 0.7141 0.842566 0.882274 2 rs2220427 0.00257131.4003 0.157434 0.117726 4 rs2220427 0.01664881 1.2682 0.201729 0.1661541 rs2634073 0.01664881 0.7885 0.798271 0.833846 3 rs2634073 0.583501560.9511 0.760807 0.769811 1 rs2723296 0.58350156 1.0514 0.239193 0.2301893 rs2723296 0.03150475 0.8367 0.661383 0.700107 2 rs2723316 0.031504751.1952 0.338617 0.299893 4 rs2723316 0.16517516 1.1172 0.622832 0.5964621 rs3853444 0.16517516 0.8951 0.377168 0.403538 3 rs3853444 0.243019260.8797 0.14121 0.157473 2 rs4576077 0.24301926 1.1367 0.85879 0.842527 4rs4576077 0.19773377 1.1482 0.161383 0.143543 1 rs6419178 0.197733770.8709 0.838617 0.856457 3 rs6419178

TABLE 4 Markers in perfect linkage disequilibrium (r² = 1.0) withrs2220427 in the CEU population in the International HapMap data set(Individuals of European descent). Also shown are correlation withsamples from Yuroba (Nigeria), and Asia (China and Japan)- cohortdescription is further documented on http://www.hapmap.org. SNP AlleleCEU_R2 CEU_frq YRI_R2 YRI_frq ASIA_R2 ASIA_frq rs17042059 1 1 0.1176470.500382 0.117647 0.473183 0.30814 rs4529121 1 1 0.116667 0.604601 0.10.539766 0.337079 rs4543199 2 1 0.116667 0.502036 0.116667 0.5397660.337079 rs10019689 1 1 0.116667 0.128175 0.341667 0.664071 0.388889rs4626276 2 1 0.116667 0.603474 0.10084 0.537439 0.333333 rs17042076 2 10.117647 0.128175 0.341667 0.664071 0.388889 rs11098089 2 1 0.1176470.549368 0.108333 0.539766 0.337079 rs11930528 4 1 0.11017 0.1207730.321429 0.662926 0.377907 rs17042098 1 1 0.116667 0.669219 0.0924370.64297 0.355556 rs17042102 1 1 0.091743 NA NA 0.580822 0.302632rs17042121 3 1 0.116667 0.736119 0.141667 0.639142 0.353933 rs10516563 31 0.109244 0.846743 0.128205 0.636329 0.364706 rs4605724 1 1 0.1166670.748252 0.083333 0.645257 0.359551 rs2350269 4 1 0.11017 0.4958060.098214 0.628257 0.346154 rs6533527 1 1 0.116667 0.425151 0.1333330.804123 0.421348 rs17042144 2 1 0.119658 0.727891 0.077586 NA NArs1906618 2 1 0.115044 NA NA NA NA rs1906617 2 1 0.116667 0.5412060.183333 0.977348 0.454545 rs12646447 2 1 0.119658 1 0.108333 1 0.444444rs12646754 4 1 0.119658 0.681842 0.11017 1 0.425 rs2129981 1 1 0.1166671 0.108333 1 0.444444 rs12639654 4 1 0.116667 0.139505 0.016667 10.438202 rs6817105 2 1 0.117647 0.27862 0.299145 1 0.440476 rs17042171 11 0.109244 0.281063 0.302521 1 0.425287 rsl 906591 1 1 0.116667 10.108333 1 0.444444 rs1906592 3 1 0.109244 0.283489 0.3 1 0.446429rs2200732 2 1 0.112069 0.272544 0.308334 1 0.449438 rs2200733 4 10.116667 0.276161 0.301724 1 0.445783 rs4611994 2 1 0.116667 0.2725440.308334 1 0.449438 rs4540107 1 1 0.116667 0.27862 0.305085 1 0.44382rs1906593 4 1 0.117647 0.285134 0.301724 1 0.438202 rsl 906596 2 10.121739 0.255864 0.330275 0.97478 0.448718

TABLE 5A. SNP markers within LD Block C04 (Between 111, 954, 811 and 112, 104, 250 on C04;NCBI Build 35; SEQ ID NO: 50). Pos in SEQ Marker ID Pos Build 35ID NO: 50 Type Strand rs1448824 111954811 1 A/G − rs1947189 111955221411 A/G − rs1947188 111955479 669 C/G − rs1992927 111956353 1543 C/T −rs1470619 111957122 2312 A/G − rs1448823 111958486 3676 A/G − rs4834327111958676 3866 A/T + rs1448822 111958702 3892 C/T − rs2044674 1119590754265 A/G − rs28445748 111959470 4660 A/T + rs2595116 111959591 4781 C/T− rs2595115 111959725 4915 A/C − rs13120244 111961948 7138 A/G +rs2723296 111962087 7277 A/G + rs2723297 111962201 7391 A/T + rs10021211111962246 7436 C/T + rs17042011 111962331 7521 C/T + rs2595114 1119627917981 C/G − rs2595113 111962792 7982 C/G − rs2595112 111963368 8558 C/G −rs6831623 111964677 9867 C/T + rs6854883 111964919 10109 C/T + rs2255793111965457 10647 A/G + rs2723298 111966089 11279 C/T + rs12505886111966218 11408 A/T + rs28718263 111966220 11410 A/T + rs12501913111966355 11545 A/C + rs13126974 111966385 11575 A/T + rs36194761111966385 11575 A/T + rs28473341 111966486 11676 C/T + rs36160675111967780 12970 G/T + rs13147139 111968764 13954 A/G + rs13147489111968795 13985 C/T + rs13147299 111968812 14002 A/C + rs13147726111968923 14113 C/T + rs13147730 111968926 14116 C/T + rs13147552111968949 14139 A/G + rs13123918 111968996 14186 A/T + rs35610510111970561 15751 C/T + rs36162200 111971480 16670 G/T + rs11098086111971997 17187 C/T + rs4034950 111972120 17310 A/G − rs11724067111972144 17334 A/G + rs2723299 111972436 17626 A/G + rs2723300111972512 17702 A/G + rs13138211 111972606 17796 A/G + rs2595075111973312 18502 C/T − rs2723301 111973731 18921 C/G + rs2595074111974709 19899 A/T − rs2723302 111974736 19926 C/G + rs2723303111974741 19931 A/G + rs2218698 111975356 20546 G/T − rs2218697111975357 20547 C/T − rs2595073 111975436 20626 A/G − rs2723307111975800 20990 A/T + rs1584430 111976043 21233 C/G − rs1584429111976151 21341 C/G − rs1900828 111976526 21716 C/T − rs7672226111976785 21975 C/T + rs1839189 111976971 22161 C/T − rs1579946111977724 22914 A/G − rs12509115 111977892 23082 A/G + rs1579945111978096 23286 A/T − rs7661383 111979181 24371 A/C + rs2122078111979201 24391 A/G − rs2122077 111979254 24444 C/T − rs2723311111979626 24816 A/G + rs7667461 111979738 24928 A/G + rs1448799111980386 25576 C/T − rs1448798 111980789 25979 C/T − rs12650829111980880 26070 A/G + rs2723312 111980956 26146 A/G + rs1900827111981343 26533 A/G − rs6815628 111981980 27170 C/T + rs6838131111981993 27183 A/G + rs6838139 111982000 27190 A/C + rs6838295111982012 27202 C/T + rs4582211 111982043 27233 A/G + rs4353966111982088 27278 A/T + rs6838536 111982144 27334 C/T + rs1375302111983068 28258 C/T + rs1375303 111983069 28259 A/G + rs7699114111983094 28284 C/T + rs2197814 111983098 28288 A/C + rs2218700111983340 28530 A/G + rs969642 111983529 28719 C/T + rs17042020111984067 29257 A/C + rs2595099 111984371 29561 A/C + rs4371683111984371 29561 A/C + rs2595093 111984960 30150 C/T − rs17625509111984998 30188 A/G + rs2723313 111985093 30283 A/G + rs2723314111985111 30301 G/T + rs2595092 111985112 30302 A/G − rs2595091111985223 30413 C/G − rs1375301 111985458 30648 A/G − rs2245595111985715 30905 C/T + rs2595088 111985958 31148 C/T − rs981150 11198623231422 C/T − rs16997168 111986643 31833 C/T + rs6812840 111986654 31844A/T + rs16997169 111986685 31875 C/T + rs4527540 111986742 31932 C/T +rs2595078 111987397 32587 A/G + rs11098087 111987538 32728 C/T +rs6843456 111988165 33355 C/T + rs998101 111988219 33409 A/G −rs13120535 111989691 34881 A/G + rs17042026 111989978 35168 A/G +rs6840960 111991045 36235 C/T + rs2122079 111991108 36298 C/T +rs2166961 111991365 36555 CIT + rs2723316 111991891 37081 C/T +rs2595079 111992019 37209 A/G + rs7665126 111992019 37209 A/G +rs2595080 111992042 37232 A/G + rs12646859 111992237 37427 G/T +rs10222783 111992430 37620 C/T + rs12498380 111992563 37753 C/T +rs2595081 111992761 37951 C/T + rs2595087 111992896 38086 G/T +rs2723317 111993104 38294 A/G + rs6419178 111993104 38294 A/G +rs13110876 111993625 38815 A/G + rs2595083 111993625 38815 A/G Irs7690164 111994069 39259 C/T i rs2595084 111994163 39353 A/G +rs2595085 111994377 39567 C/G + rs2595086 111994385 39575 C/T +rs2723318 111994576 39766 G/T + rs17042050 111994805 39995 C/T +rs9998222 111995088 40278 A/G + rs2723319 111995233 40423 A/T +rs2595087 111995380 40570 C/T + rs17042052 111995521 40711 A/T +rs28558677 111995664 40854 G/T + rs6817731 111995691 40881 A/C +rs2723320 111997050 42240 C/T + rs12644107 111997588 42778 C/T +rs28482179 111998237 43427 C/T + rs28759131 111998559 43749 C/T +rs1448817 111998657 43847 A/G + rs28526075 111998725 43915 A/G +rs17042059 111998790 43980 A/G + rs10014075 112000023 45213 G/T +rs10026140 112000455 45645 G/T + rs13351232 112000455 45645 G/T +rs7666806 112000477 45667 G/T + rs10028327 112000489 45679 G/T +rs12650941 112002415 47605 A/T + rs28650220 112002617 47807 C/T +rs13113361 112002671 47861 G/T + rs13113522 112002686 47876 G/T +rs4529121 112003159 48349 A/G + rs6831284 112003582 48777 G/T +rs10009621 112003846 49036 C/T + rs10021534 112003945 49135 C/T +rs10032150 112004222 49412 A/G + rs10024267 112004571 49761 C/T +rs10012705 112004726 49916 C/T + rs11943627 112005073 50263 C/T +rs4543199 112005744 50934 C/T + rs28410055 112006340 51530 A/G +rs7693227 112006532 51722 C/T + rs6852197 112006679 51869 A/G +rs12647316 112006855 52045 C/T + rs12647393 112006886 52076 G/T +rs10019645 112007248 52438 G/T + rs10019689 112007473 52663 A/C +rs4626276 112007593 52783 A/C + rs10022067 112007672 52862 C/T +rs4469143 112007678 52868 C/G + rs6836206 112007902 53092 C/T +rs13150693 112008086 53276 G/T + rs11737637 112008416 53606 C/T +rs5011975 112008427 53617 A/G + rs6811511 112008429 53619 A/C +rs4383676 112008437 53627 A/G + rs28392642 112009161 54351 C/T +rs17631468 112009386 54576 A/G + rs17042076 112009942 55132 C/T +rs4434326 112010480 55670 C/T + rs17042081 112010815 56005 G/T +rs4833436 112011350 56540 C/T + rs7679623 112011519 56709 A/C +rs11098088 112011728 56918 C/T + rs4530699 112011761 56951 A/T +rs11098089 112011830 57020 A/C + rs17042088 112012418 57608 C/T +rs12648785 112013496 58686 A/G + rs12639820 112013644 58834 C/T +rs10001807 112013708 58898 A/G + rs10024486 112013722 58912 G/T +rs12648889 112013890 59080 C/G + rs28376747 112013925 59115 A/G +rs11098090 112014012 59202 C/T + rs11944778 112014571 59761 A/G +rs7436333 112014951 60141 A/C + rs4307025 112015107 60297 A/T +rs4447925 112015252 60442 C/T + rs28523292 112015772 60962 C/T +rs28635581 112015858 61048 C/T + rs28508237 112016004 61194 C/T +rs28521134 112016167 61357 C/T + rs17042093 112017716 62906 C/G +rs11930438 112017749 62939 C/T + rs28542185 112017795 62985 C/T +rs11930528 112017798 62988 G/T + rs13121382 112020177 65367 G/T +rs7439625 112021082 66272 A/T + rs28501998 112021318 66508 A/T +rs10016838 112021718 66908 C/T + rs17042098 112021762 66952 A/G +rs10005076 112021953 67143 C/T + rs10027473 112022056 67246 A/G +rs2634073 112023387 68577 A/G − rs1906611 112023520 68710 A/G −rs1906610 112023521 68711 C/T − rs28446238 112024025 69215 A/C +rs1906609 112024055 69245 A/C − rs34916665 112025294 70484 G/T +rs17042102 112026230 71420 A/G + rs17042104 112026555 71745 C/T +rs10015819 112026628 71818 C/T + rs2634071 112026824 72014 A/G −rs10007386 112028021 73211 C/T + rs10007547 112028050 73240 A/G +rs12647522 112028465 73655 C/T + rs1906614 112028900 74090 A/G −rs2723335 112029230 74420 A/G + rs17042112 112029281 74471 C/T +rs17042115 112029414 74604 A/G + rs10013510 112029527 74717 C/G +rs11939057 112029755 74945 C/T + rs2634076 112029877 75067 A/G −rs2723293 112031377 76567 A/G + rs28494131 112032777 77967 C/T +rs2634075 112033582 78772 A/G − rs13121715 112034239 79429 G/T +rs2634074 112034645 79835 A/T − rs17042121 112034705 79895 A/G +rs10516563 112035326 80516 G/T + rs17042125 112035883 81073 A/G +rs13136439 112036254 81444 G/T + rs13114686 112036503 81693 C/T +rs36166388 112037782 82972 A/G + rs2450934 112038532 83722 A/C −rs36138049 112040474 85664 A/C + rs36168695 112040495 85685 A/G +rs36129850 112040548 85738 A/G + rs12513264 112041966 87156 C/T +rs2882365 112041975 87165 A/G + rs36139649 112042072 87262 C/T +rs4033107 112042072 87262 C/T + rs36176419 112042092 87282 A/G +rs4033108 112042092 87282 A/G + rs4450997 112042160 87350 A/C +rs2350268 112042213 87403 A/G + rs4613627 112042225 87415 A/C +rs4033109 112042227 87417 C/T + rs4833443 112042247 87437 C/T +rs2723336 112042258 87448 C/T − rs4033111 112042302 87492 A/G +rs1807360 112042333 87523 C/T − rs4605724 112042685 87875 A/C +rs6856879 112043066 88256 A/T + rs6834418 112043172 88362 C/T +rs2466455 112043219 88409 A/G − rs6857810 112043220 88410 A/G +rs2634079 112043541 88731 C/G − rs28366840 112043710 88900 A/T +rs2350269 112044728 89918 C/T + rs7665409 112045070 90260 C/T +rs6533527 112045118 90308 A/C + rs12649717 112045283 90473 A/C +rs6822831 112045374 90564 A/G + rs35916701 112046074 91264 C/T +rs6829419 112046178 91368 C/T + rs2723334 112046356 91546 A/G −rs2634078 112046528 91718 C/T − rs12512819 112046597 91787 C/T +rs17042144 112047270 92460 C/T + rs2171594 112047908 93098 A/G −rs6842887 112048170 93360 A/G + rs2171593 112048375 93565 G/T −rs7690874 112048681 93871 A/G + rs17042145 112049101 94291 G/T +rs17042146 112049106 94296 C/T + rs9998815 112049904 95094 C/G +rs7683336 112051207 96397 C/T + rs17042150 112051452 96642 A/T +rs10016842 112051810 97000 C/T + rs10005432 112052219 97409 A/G +rs1906620 112052624 97814 C/T − rs1906619 112052670 97860 C/T −rs1906618 112053026 98216 C/T − rs1906617 112053418 98608 C/T −rs6847935 112054255 99445 A/T + rs6831873 112055138 100328 C/T +rs1906616 112055172 100362 C/T − rs6837901 112055712 100902 C/T +rs2723333 112056695 101885 C/T − rs12646447 112056930 102120 C/T +rs6820568 112057435 102625 C/T + rs1906615 112059402 104592 A/C −rs2634077 112061112 106302 A/G − rs7689774 112061114 106304 G/T +rs12646754 112061176 106366 C/T + rs35807830 112061497 106687 G/T +rs2129983 112061684 106874 C/T − rs2129982 112061747 106937 C/T −rs2129981 112061803 106993 A/C − rs6854111 112062140 107330 A/T +rs12639654 112062899 108089 C/T + rs4515229 112062985 108175 A/G +rs2129984 112063010 108200 C/T + rs6817105 112063372 108562 C/T +rs12503217 1120G3765 108955 C/T + rs2634070 112064016 109206 A/C +rs17042171 112065891 111081 A/C + rs7434417 112066042 111232 A/G +rs1906591 112066493 111683 A/G + rs1906592 112066608 111798 G/T +rs12510087 112066632 111822 A/G + rs7661554 112067221 112411 A/G +rs34796144 112067333 112523 A/C + rs2200732 112067646 112836 C/T +rs2200733 112067773 112963 C/T + rs17042175 112068571 113761 A/T +rs4611994 112068645 113835 C/T + rs4540107 112068706 113896 A/C +rs1906593 112069526 114716 C/T + rs4371684 112069651 114841 A/G +rs1906594 112069739 114929 A/G + rs1906595 112069788 114978 G/T +rs1906596 112069840 115030 C/T + rs6838775 112069908 115098 G/T +rs2129977 112070036 115226 A/G + rs2129978 112070158 115348 A/C +rs1906597 112070190 115380 G/T + rs1906598 112070229 115419 C/T +rs1906599 112070290 115480 C/T + rs1906600 112070480 115670 C/T +rs1906601 112070883 116073 C/T + rs1906602 112070927 116117 C/T +rs1906603 112071040 116230 C/T + rs28645285 112071426 116616 A/G +rs2171590 112071435 116625 C/T + rs6852357 112071939 117129 C/T +rs13143308 112072023 117213 G/T + rs2220427 112072493 117683 C/T +rs17632693 112072538 117728 C/T + rs11935917 112072850 118040 A/G +rs4833456 112073911 119101 C/T + rs12644625 112074117 119307 C/T +rs4400058 112074277 119467 A/G + rs1906604 112074452 119642 A/G +rs1906605 112074796 119986 C/T + rs13126975 112075129 120319 A/T +rs6837490 112075447 120637 C/T + rs6843082 112075671 120861 A/G +rs13105878 112075751 120941 A/C + rs6533528 112076843 122033 A/G +rs7692272 112076857 122047 G/T + rs2171591 112077012 122202 A/G +rs17042195 112077142 122332 C/G + rs11931959 112077289 122479 A/G +rs17042198 112077582 122772 G/T + rs10033464 112078365 123555 G/T +rs2171592 112078392 123582 C/T + rs13121924 112078423 123613 A/G +rs2129979 112078601 123791 G/T + rs2350539 112078814 124004 G/T +rs1906606 112080996 126186 A/C + rs7672570 112081189 126379 C/T +rs4834418 112081408 126598 A/G + rs723364 112082075 127265 C/G −rs723363 112082105 127295 A/G − rs7697491 112083422 128612 A/T +rs13125644 112083505 128695 A/G + rs2350294 112084449 129639 A/G −rs2350293 112084451 129641 A/G − rs3855819 112084767 129957 C/G −rs2220428 112085064 130254 A/G + rs2220429 112085089 130279 A/C +rs11727566 112085934 131124 A/T + rs13141190 112086218 131408 A/G +rs4032976 112086371 131561 A/G − rs3866829 112086379 131569 A/C −rs7671348 112086408 131598 A/G + rs3866830 112086598 131788 C/G −rs6811267 112086942 132132 C/T + rs3853440 112087213 132403 C/T −rs3853441 112087344 132534 A/G − rs3853442 112087632 132822 C/T −rs3853443 112087733 132923 A/G − rs4124158 112087798 132988 C/T +rs4124159 112087847 133037 A/G + rs12506083 112088016 133206 A/C +rs34809282 112088051 133241 A/G + rs7683219 112088051 133241 A/G +rs7683618 112088259 133449 A/C + rs7683625 112088269 133459 A/G +rs7662050 112088325 133515 C/T + rs36183416 112088804 133994 C/T +rs4447926 112088804 133994 C/T + rs4594787 112088813 134003 A/G +rs10390275 112089009 134199 A/T + rs36179422 112089009 134199 A/T +rs10006659 112089030 134220 G/T + rs36181695 112089078 134268 A/G +rs7440730 112089078 134268 A/G + rs10006881 112089277 134467 C/T +rs36149087 112089277 134467 C/T + rs6533530 112089540 134730 C/T +rs6533531 112089569 134759 G/T + rs3866831 112089718 134908 C/T −rs4269241 112089833 135023 A/G + rs4032975 112089842 135032 A/C −rs4032974 112090140 135330 A/G − rs4124160 112090452 135642 A/G +rs3866832 112091304 136494 C/G − rs3853444 112091740 136930 A/G −rs7662345 112091766 136956 A/G + rs2350545 112092258 137448 C/T −rs17042215 112092562 137752 C/T + rs2003121 112093023 138213 C/T +rs880309 112093143 138333 A/G + rs9991046 112093346 138536 G/T +rs17042216 112094463 139653 C/T + rs17570669 112094486 139676 A/T +rs17042218 112094520 139710 A/G + rs17042223 112094922 140112 C/T +rs3866833 112095138 140328 C/T − rs17042224 112096509 141699 G/T +rs13130446 112096760 141950 C/T + rs10516564 112096896 142086 A/G +rs7686320 112097215 142405 A/T + rs7686499 112097282 142472 C/T +rs17042230 112097319 142509 C/T + rs4124161 112097459 142649 C/T +rs4576077 112098061 143251 C/T + rs4260600 112098098 143288 C/T +rs12644093 112098445 143635 A/G + rs4124162 112098593 143783 A/G +rs7674295 112099042 144232 A/G + rs11938968 112100356 145546 A/G +rs28601812 112101457 146647 A/C + rs4032983 112101551 146741 G/T +rs3866834 112101617 146807 A/G + rs6852021 112101716 146906 A/G +rs28580491 112102583 147773 C/T + rs13110989 112102671 147861 G/T +rs3866835 112102983 148173 C/T − rs4124163 112103203 148393 A/G +rs3866836 112103244 148434 A/G + rs17042238 112103458 148648 A/G +rs4124164 112104250 149440 C/T +B. Microsatellite markers within LD Block C04 (Between 111, 954, 811 and 112,104, 250 on C04; NCBI Build 35; SEQ ID NO: 50). End MarkerStart position position Forward primer Reverse Primer D4S193 112062811112062911 ACAACCCCATTTGTGAAGAC TTTATAGAAAATTTAGCATGGA D4S2940 112070055112070267 CTAAGTTGTGCAGCCATGAA TGGAACCACTTTTGCAGTAA D4S406 112076047112076292 CTGGTTTTAAGGCATGTTTG TCCTCAGGGAGGTCTAATCA

TABLE 6 Key to sequences presented in sequence listing. SEQ ID NO MarkerID 1 rs2220427 2 rs17042059 3 rs4529121 4 rs4543199 5 rs10019689 6rs4626276 7 rs17042076 8 rs11098089 9 rs11930528 10 rs17042098 11rs17042102 12 rs17042121 13 rs10516563 14 rs4605724 15 rs2350269 16rs6533527 17 rs17042144 18 rs1906618 19 rs1906617 20 rs12646447 21rs12646754 22 rs2129981 23 rs12639654 24 rs6817105 25 rs17042171 26rs1906591 27 rs2200732 28 rs2200733 29 rs4611994 30 rs4540107 31rs1906593 32 rs1906596 33 rs2634073 34 rs1906592 35 rs2723296 36rs16997168 37 rs2723316 38 rs6419178 39 rs1448817 40 rs13105878 41rs10033464 42 rs13141190 43 rs3853444 44 rs4576077 45 D4S406 46rs7668322 47 rs2197815 48 rs6831623 49 rs2595110 50 LD Block C04 51rs13143308

Example 2 Characterization of AF Risk Variants

The following contains further description of the identification ofvariants conferring risk for atrial fibrillation on chromosome 4q25

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmiain man and is characterized by chaotic electrical activity of theatria¹. It affects one in ten individuals over eighty, causessignificant morbidity, and is an independent predictor of mortality².Recent studies have provided evidence of a genetic contribution toAF³⁻⁵. Mutations in potassium channel genes have been associated withfamilial AF⁶⁻¹⁰ but account for only a small fraction of all AFcases^(11,12). We performed a genome-wide association scan, followed byreplication studies in three populations of European descent and aChinese population from Hong Kong and find a strong association betweentwo sequence variants on chromosome 4q25 to AF. Approximately 35% ofindividuals of European descent have at least one of the variants andthe risk of AF increases by 1.72 and 1.39 per copy. The association tothe stronger variant was replicated in the Chinese population, where itis carried by 75% of individuals and risk of AF is increased by 1.42 percopy. A stronger association was observed in individuals with typicalatrial flutter (AFI). Both variants are adjacent to PITX2, which isknown to play a critical role in left-right asymmetry of the heart¹³⁻¹⁵.We conducted a genome-wide association study using the Illumina Hap300BeadChip on an Icelandic population with AF and/or AFI. 316,515 SNPssatisfying our quality criteria were tested individually for associationto AF/AFI in a sample of 550 patients and 4,476 controls from Iceland.Three strongly correlated SNPs, all located within a single linkagedisequilibrium (LD) block on chromosome 4q25, were the only SNPs foundto be genome-wide significant after accounting for the 316,515 SNPstested (P<0.05/316,515=1.58×10⁻⁷): rs2200733 (OR=1.75; P=1.6×10⁻¹⁹),rs2220427 (OR=1.75; P=1.9×10⁻¹⁰) and rs2634073 (OR=1.60; P=2.1×10⁻⁹).These results and all other results based on the Icelandic populationwere adjusted for the relatedness of individuals. The two mostsignificant SNPs, rs2200733 and rs2220427, are perfect proxies for oneanother in the CEPH CEU HapMap¹⁶ dataset and are close to being perfectproxies for one another in the Icelandic dataset (D′=1, r²=0.999),therefore, only rs2200733 will be referred to in the followingdiscussion. The correlation of rs2634073 to rs2200733 is weaker in theIcelandic dataset (D′=0.95, r²=0.605). Upon further study of theIllumina Hap300 SNPs in the vicinity of the first three SNPs andconditioning on the association to rs2200733, an association to a newSNP, rs10033464, was identified (OR=1.42; P=0.0024). After accountingfor the association to rs2200733 and rs10033464, the association tors2634073 was no longer significant (P=0.30). Henceforth, allassociation results for rs2200733 T and rs10033464 T, including thosepresented in Table 7, are based on comparison to the wild type haplotypewhich carries neither of the two at risk alleles, rather than comparisonto the major alleles of each SNP separately. Specifically, odds-ratiosfor rs2200733 T and rs10033464 T are each computed conditionally andcould be interpreted as the estimated relative risk of each variantcompared to the wild-type. The at risk alleles T of rs2200733 and T ofrs10033464 have estimated population allelic frequencies of 12.05% and8.53% in Iceland, respectively, and are never observed together on thesame chromosome, in the Icelandic dataset or in the CEU HapMap dataset.A third SNP, rs13143308, which has a minor allele that correspondscompletely to chromosomes carrying either the T allele of rs2200733 orthe T allele of rs10033464, was identified through the CEU HapMapdataset. FIG. 2 demonstrates the haplotype structure over the key SNPsof the associated region. Sets of SNPs, that are perfect proxies (i.e.,perfect surrogates, r2=1.0 to the tagging SNP) of each of these threekey SNPs in the CEU HapMap samples, are provided in Table 9 and relativelocations displayed in FIG. 3. We emphasize that the SNPs named shouldbe considered representatives of the haplotypes defined by the SNPswhich they are equivalent to and are primarily chosen for the sake ofconvenience.

A microsatellite marker, D4S406, located in the same LD block as the twoSNPs was identified. In Iceland, three of the four shortest alleles ofD4S406 (-8, -4, and -2) combine to form a near perfect surrogate for theT allele of rs2200733 (D′=0.995, r²=0.98) and the two shortest remainingalleles (-6 and 0) form a good surrogate of the T allele of rs10033464(D′=0.98, r²=0.75) (Table 10). None of the remaining (longer) alleles ofD4S406 are associated to AF/AFI after accounting for the effect of theshort alleles. For the replication of the original observation inIceland the D4S406 genotypes were used to provide information when SNPgenotypes were not available.

In an attempt to replicate our original discovery we analyzed anadditional Icelandic samples consisting of 2,251 AF/AFI patients and13,238 controls (Table 7). The association of both SNPs to AF/AFI wasreplicated in these samples (OR=1.64, P=2.7×10⁻²³ for rs2200733,OR=1.40, P=8.2×10⁻⁸ for rs10033464) and both achieve genome-widesignificance in the combined Icelandic samples (OR=1.68, P=1.9×10⁻³⁰ forrs2200733, OR=1.38, P=9.4×10⁻⁹ for rs10033464). We also typed all the 18Hap300 Illumina SNPs in the region around our signal in 404 of theadditional AF cases and 2,036 of the additional controls. None of theseSNPs remained significant after accounting for the association tors2200733 and rs10033464 (Table 11).

In further attempts to replicate our results, we tested these variantsfor an association to AF in two populations of European ancestry, onefrom Sweden, consisting of 143 cases and 738 controls, and the otherfrom the United States (U.S.), consisting of 636 cases and 804 controls(Table 7). The association to rs2200733 was strongly replicated in bothpopulations (OR=2.01, P=0.00027 in Sweden, OR=1.84, P=9.8×10⁻¹⁰ in theU.S.). The association to rs10033464 is weaker, but was nonethelessreplicated in the Swedish population (OR=1.65, P=0.0087) and was nearlysignificant in the U.S. population (OR=1.30, P=0.052). When combinedwith the Icelandic samples, the association to rs2200733 was unequivocal(OR=1.72, P=3.3×10⁻⁴¹), and the significance of rs10033464 was wellbeyond the threshold of genome-wide significance (OR=1.39, P=6.9×10⁻¹¹).Assuming the multiplicative model, the population attributable risk(PAR) of the two variants combined is approximately 20% in populationsof European ancestry.

Finally, we attempted to replicate these signals in a Han Chinesepopulation from Hong Kong consisting of 333 cases and 2,836 controls.The association to rs2200733 T was significantly replicated (OR=1.42,P=0.00064), but the association to rs10033464 T was not significant,although in the right direction (OR=1.08, P=0.55) (Table 7).Interestingly, the T allele of rs2200733 is much more frequent in theChinese (allelic frequency in controls: 0.528) than in those of Europeandescent (allelic frequency in controls: 0.098-0.139) (FIG. 2) which isreflected in a greater joint PAR of approximately 35%, even though theestimated risk is less. The LD block containing the two variants is morefragmented in the Chinese CHB and Japanese JPT HapMap samples than inthe CEU HapMap samples (FIG. 3). We therefore analysed several markersin the Hong Kong population which were in perfect LD with rs2200733 inthe CEU samples, but in imperfect LD in the CHB and JPT samples (Table12). These markers had weaker apparent association to AF than rs2200733,suggesting that the functional variants driving the association islocated in the approximately 20 kb region around the original rs2200733variant and defined by the SNPs that remain equivalent to rs2200733 inthe CHB and JPT samples (coloured red in FIG. 3).

For the initial Icelandic discovery samples, rs2200733 had asignificantly higher OR than rs10033464 (P=0.041). This held true in thereplication samples, and overall there is a significant difference inthe risks associated with the two variants (P=0.00019 in the combinedEuropean samples and P=0.0099 in Hong Kong). When genotype-specific oddsratios were studied, some deviation away from the multiplicative modelis detectable in the combined dataset (P=0.018 for European samples, seeTable 13). Estimated risks of heterozygous carriers relative tonon-carriers were similar, but homozygous carriers of rs2200733 T andrs10033464 T have estimated risks that were, respectively, higher andlower than that predicted by a multiplicative model. A similar trend wasseen in the Hong Kong samples; although the sample size is too small tohave power to detect such deviations with significance. In the combinedpopulations of European descent the observed OR for individualshomozygous for rs2200733 T was 3.64 as compared to individualshomozygous for the wild type haplotype and 1.77 for the Chinesepopulation demonstrating that these variants are important components inany predictive modeling of AF.

The age at diagnosis of AF/AFI for the Icelandic samples correlates withthe two SNPs (diagnosis occurs 2.28 years earlier per T allele ofrs2200733 and 1.10 years earlier per T allele of rs10033464, jointP=1.29×10⁻⁶). The effect of the age at diagnosis was also evaluated bymeasuring the strength of association while stratifying by age atdiagnosis. The association of the two variants is strongest in thosediagnosed at a younger age, although the risk remains significant evenin those diagnosed after reaching 80 years of age (Table 8). Informationon age at diagnosis of AF was not available for the Swedish samples. TheU.S. samples were comprised of two main groups, younger patients witheither lone AF or AF and hypertension (HTN), and older AF cases who aremostly hemorrhagic and ischemic stroke patients. In both populationsthere is a clear trend towards a stronger association in younger AFcases than in older cases. Our analysis of the data did not suggest anydifferential association by sex (Table 8).

AF1 often accompanies AF, but can occur in isolation¹⁷. Interestingly weobserved a strong association between the variants and the small subset(N=116) of the AF1 Icelandic patients (OR=2.60, 95% confidence interval(CI)=1.83-3.68, P=7.5×10⁻⁸ for rs2200733, OR=1.94, 95% CI=1.26-3.00,P=0.0028 for rs10033464). Indeed, for rs2200733, the OR for thesedefinite AFI cases is significantly higher than that for the cases withan AF phenotype (P=0.0026), and close to significantly higher forrs10033464 (P=0.084). Our results suggest that while these traits sharegenetic risk factors, AFI is less influenced by phenocopies than AF.

Neither variant showed a association to obesity, hypertension ormyocardial infarction in the Icelandic samples, all known risk factorsfor AF (observed OR<1.1 in all instances, Table 14). Although thesenegative results do not exclude the possibility that the new variantsassociate with these phenotypes, they do suggest, along with the highrisk in U.S. lone AF and earlier age at onset in carriers, that the newvariants are not affecting risk of AF through these known risk factors.

There is no known gene present in the LD block containing rs2200733 andrs10033464 (FIG. 3). The LD block contains one spliced EST (DA725631)and two single-exon ESTs (DB324364 and AF017091). RT-PCR of cDNAlibraries from various tissues did not detect the expression of theseESTs (Table 16). The PITX2 gene located in the adjacent upstream LDblock is the gene closest to the risk variants. Several markers withinthe LD block containing the PITX2 gene are correlated to the markersshowing association to AF and Afl, as shown in Table 18. The proteinencoded by this gene, the paired-like homeodomain transcription factor2, is an interesting candidate for AF/AFI as it is known to play animportant role in cardiac development by directing asymmetricmorphogenesis of the heart¹³. In a mouse knockout model Pitx2 was shownto suppress a default pathway for sinoatrial node formation in the leftatrium^(14,15). There is very little mRNA expression of PITX2 in alleasily accessible tissues, such as blood and adipose tissue, hamperingthe study of correlation between genotypes and expression levels. Thenext gene upstream of PITX2 is ENPEP, an aminopeptidase responsible forthe breakdown of angiotensin II in the vascular endothelium¹⁸. This geneis expressed more widely, but the variants associated with AF showed nocorrelation to its expression in blood or adipose tissue. No otherannotated genes are located within a 400 kb region upstream and 1.5 Mbregions downstream of the associated variants.

In summary, we have identified two variants on chromosome 4q25 that arestrongly associated with AF in three distinct populations of Europeandescent. The stronger variant also replicates well in a Chinesepopulation where it is much more common and has higher PAR than inpopulations of European descent. This association is particularlycompelling in younger patients and in those with lone AF, but is alsopresent in older patients with more commonly encountered forms of AF.Although the mechanism for this association is unknown, our resultsprovide a foundation for further studies on the molecular underpinningsof AF.

Methods Subjects

The Icelandic cases consisted of all patients diagnosed with AF and/orAFI at the two largest hospitals in the country from 1987 to 2005. TheSwedish cases were recruited from 1996 to 2002 as a part of an ongoinggenetic epidemiology study, the South Stockholm Ischemic Stroke Study.The U.S. cases were a mixture of stroke patients with a AF diagnosis andyounger consecutive patients with lone AF or AF with a coexistingdiagnosis of hypertension. The Hong Kong cases were a collection ofstroke and diabetes patients with an AF diagnosis. The AF diagnosis wasconfirmed by a twelve lead electrocardiogram in all study populations.

The Icelandic controls were chosen at random from individuals who haveparticipated in other genetic studies at deCODE, excluding first-degreerelatives of patients and controls (Table 15). The Swedish controls wererecruited from the same region as patients from blood donors (in 2001)and healthy volunteers (1990-1994). The U.S. controls were recruitedfrom a large primary care practice and from patients participating in ahemorrhagic stroke study. The Hong Kong controls were individualswithout an AF diagnosis.

Icelandic Study Population

This study initially included the all patients consenting toparticipation, which were diagnosed with AF and/or AFI (ICD 10 diagnosis148 and ICD 9 diagnosis 427.3) at Landspitali University Hospital inReykjavik, the only tertiary referral centre in Iceland, and at AkureyriRegional Hospital, the second largest hospital in the country, from 1987to 2005. All diagnoses were confirmed by a twelve lead electrocardiogram(EKG) which was manually read by a cardiologist. All cases wereincluded, regardless of whether the patients had clinical symptoms ornot, except those diagnosed only immediately after open cardiac surgery.

A set of 550 cases were successfully genotyped according to our qualitycontrol criteria in a genome-wide SNP genotyping effort, using theInfinium II assay method and the Sentrix HumanHap300 BeadChip (Illumina,San Diego, Calif., USA). The mean age at diagnosis for this initialgroup of 550 patients (370 males and 180 females) was 72.5 (SD=11.0)years and the range was from 34.7-96.2 years. The validation group of2,273 patients (1,359 males and 913 females) had a mean age at diagnosisof 70.5 (SD=13.0) and the range was from 16.8-100.6. The AF/AFI freecontrols (2,201 males and 2,275 females at the initial genome-widescreening with mean age 61.5 (SD=15.8) and 5,654 males and 7,597 femalesat the validation stage with mean age 61.9 (SD=18.4)) used in this studyconsisted of controls randomly selected from the Icelandic genealogicaldatabase and individuals from other ongoing related genetic studies atdeCODE. Controls having first-degree relatives (siblings, parents oroffspring) with AF/AFI, or a first-degree control relative, wereexcluded from the analysis.

Icelandic MI, Obesity and Hypertension Populations

Individuals who suffered an MI were identified from a registry of over10,000 individuals who: a) had an MI before the age of 75 in Iceland inthe years 1981 to 2002 and satisfy the MONICA criteria 9 (REF II), orhad MI discharge diagnosis from the major hospitals in Reykjavik in theyears 2003 and 2005. MI diagnoses of all individuals in the registryfollow strict diagnostic criteria based on signs, symptoms,electrocardiograms, cardiac enzymes and necropsy findings. Genotypeinformation was available for 2,462 males and 1,114 females, mean age72.6 (SD=11.7). Body mass index (BMI) was measured for individualsparticipating in the cardiovascular atrial fibrillation and/or stroke(CVD) genetics program at deCODE (either patients with CVD, their firstdegree relatives or spouses). For the purpose of this study subjectswith BMI>35 were defined as obese. Genotype information was availablefor 555 males and 1,046 females, mean age 53.2 (SD=16.1). Hypertensivepatients included those who had attended the ambulatory hypertensionclinic at the Landspitali, University Hospital in Iceland and/or hadbeen given the diagnosis on discharge from the hospital. The diagnosiswas verified by confirming that they were takingantihypertensivemedications as a treatment for hypertension. Genotypeinformation was available for 1,293 males and 1,327 females, mean age71.5 (SD=12.5). The study was approved by the Data Protection Commissionof Iceland and the National Bioethics Committee of Iceland. Writteninformed consent was obtained from all patients, relatives and controls.

Swedish Study Population

Patients with ischemic stroke or TIA attending the stroke unit or thestroke outpatient clinic at Karolinska University Hospital, Huddingeunit in Stockholm, Sweden were recruited from 1996 to 2002 as part of anongoing genetic epidemiology study, the South Stockholm Ischemic StrokeStudy (SSISS). The study was approved by the Bioethics Committee ofKarolinska Institutet (Dnr 286/96 and 08/02). AF diagnosis in theSwedish samples was based on a twelve lead EKG. The fraction of males inthe Swedish AF cases was 46.2% and the mean age at stroke diagnosis forthe Swedish AF cases was 74.4 (SD=8.7).

The Swedish controls used in this study are population-based controlsrecruited from the same region in central Sweden as the patients,representing the general population in this area. The individuals wereeither blood donors (recruited in 2001) or healthy volunteers (collectedin 1990-1994) recruited by the clinical chemistry department at theKarolinska University Hospital to represent a normal referencepopulation. The fraction of males in the Swedish controls was 59.7% andthe mean age at recruitment for the Swedish controls was 43.1 (SD=12.3).

U.S. Study Population

U.S. subjects were enrolled in ongoing case-control and cohort studiesat Massachusetts General Hospital (MGH) between January 1998 and July2006. All aspects of these studies have been approved by the localInstitutional Review Board. Subjects enrolled in the case-control studyconsisted of patients hospitalized with acute ischemic or hemorrhagicstroke confirmed by CT or MRI, admitted to a single acute care hospital.Of the 328 hemorrhagic stroke patients recruited 78 were diagnosed withAF and were used as cases for the current study, the remaining 250 wereused as controls. 170 ischemic stroke patients had an AF diagnosis andwere treated as cases but no ischemic stroke patients were treated ascontrols. Patients were excluded for primary subarachnoid hemorrhage andfor intracerebral hemorrhage secondary to head trauma, tumor, vascularmalformation, or vasculitis. 624 stroke-free controls were recruitedfrom a large, primary care practice (>18 000 patients) serving thehospital catchment area as well as the hospital's AnticoagulationManagement Service. 70 of the 624 individuals collected as controls werediagnosed with AF and treated cases for the purposes of the currentstudy. 50.9% of all individuals used as controls were males and theirmean age was 67.4 (SD=12.3). All subjects or an accompanying informantprovided informed consent for participation in genetic studies and wereinterviewed prospectively regarding medical history, medications, socialand family history. Presence or absence of atrial fibrillation wasprospectively documented through interview and from review of medicalrecords.

The second part of the U.S. subjects consisted of consecutive patientswith lone AF or AF with coexisting diagnosis of hypertension referred tothe arrhythmia service who provided written informed consent forparticipation in genetic. Inclusion criteria were AF documented by EKG,and an age less than or equal to 65 years. The exclusion criteria werestructural heart atrial fibrillation and/or stroke as assessed byechocardiography, rheumatic heart atrial fibrillation and/or stroke,hyperthyroidism, myocardial infarction, or congestive heart failure.Each patient underwent a physical examination and a standardizedinterview to identify past medical conditions, medications, symptoms andpossible triggers for initiation of AF. All patients were evaluated bytwelve lead EKG, echocardiogram, and laboratory studies. EKGs andechocardiograms were interpreted using standard criteria.

Hong Kong Study Population

All subjects in the Hong Kong study population were of southern HanChinese ancestry residing in Hong Kong. The cases consisted of 217individuals (49.1% male, mean age 68.1 (SD=9.6)) selected from thePrince of Wales Hospital Diabetes Registry²³ and 116 subjects (30.2%male, mean age 76.1 (SD=10.9)) from the Stroke Registry²⁴. All subjectswere diagnosed to have atrial fibrillation by EKG. The controlsconsisted of 2,836 subjects without evidence of AF. Informed consent wasobtained for each participating subject. This study was approved by theClinical Research Ethics Committee of the Chinese University of HongKong.

Illumine Genome-Wide Genotyping

All Icelandic case- and control-samples were assayed with the InfiniumHumanHap300 SNP chips (Illumina, SanDiego, Calif., USA), containing 317503 haplotype tagging SNPs derived from phase I of the InternationalHapMap project. Of the SNPs assayed on the chip, 162 SNPs generated nogenotypes, and an additional 178 SNPs had yield lower than 90%.Forty-eight SNPs were monomorphic and 107 others nearly monomorphic(i.e. the minor allele frequency in the combined cohort of patients andcontrols was less than 0.001). An additional 475 SNPs showed verysignificant distortion from Hardy-Weinberg equilibrium in the controls(p<1×10⁻¹⁰). Lastly, a few markers (n=18) were determined to havegenotyping problems after investigation of particular regions andpossible signals in several different on-going genome-wide associationstudies in house. Thus, the final analyses presented in the textutilizes 316,515 SNPs. Any samples with a call rate below 98% wereexcluded from the analysis.

Single SNP- and Microsatellite Genotyping.

SNP genotyping was carried out by the Centaurus (Nanogen) platform²⁵.The quality of each Centaurus SNP assay was evaluated by genotyping eachassay in the CEU and/or YRI HapMap samples and comparing the resultswith the HapMap data. Assays with >1.5% mismatch rate were not used anda linkage disequilibrium (LD) test was used for markers known to be inLD.

Association Analysis

An attempt was made to genotype all participating individuals forrs2200733, rs4611994 (a perfect proxy for rs2200733), rs13143308, andrs6843082 (a perfect proxy for rs13143308). For each of the SNPs, yieldwas higher than 90% in every group. In addition genotypes for the D4S406microsatellite were available for all Icelandic and Swedish subjects.Because of the redundancy in genotyping, observed genotypes reduced theamount of information lost due to missing genotypes through a likelihoodapproach we have used before²⁶. This ensured that results presented inthe tables were always based on the same number of individuals, allowingmeaningful comparisons of results. As data on rs10033464 was onlydirectly available in the initial Icelandic discovery samples and in theHapMap project the rs2200733 C rs13143308 T haplotype was used to tagthis SNP. This tagging was perfect in both the initial discovery samplesand the CEPH CEU HapMap samples.

A likelihood procedure described in a previous, and implemented in theNEMO software, was used for the association analyses. We tested theassociation of an allele to each phenotype using a standard likelihoodratio statistic, which, if the subjects were unrelated, would haveasymptotically a chi-square distribution, with one degree of freedom,under the null hypothesis. Allele-specific OR was calculated assuming amultiplicative model for the two chromosomes of an individual⁴. Resultsfrom multiple case-control groups were combined using a Mantel-Haenszelmodel⁵ in which the groups were allowed to have different allelicpopulation frequencies, haplotypes and genotypes but were assumed tohave common relative risks. There was no significant deviation fromHardy-Weinberg equilibrium (HWE) in any control group.

In Tables 7 and 8, P values for both rs2200733 and rs10033464 werecomputed based on comparison to the wild type rs2200733 C, rs13143308 G,rs10033464 G haplotype carrying neither of the at risk alleles. Thecorresponding conditional odds ratio for rs2200733 T is defined as[f(rs2200733 T)/f(WT)]/[p(rs2200733 T)/p(WT)] where WT denotes thewild-type haplotype, and f(•) and p(•) denote frequencies in cases andcontrols respectively. Under the multiplicative model and when thecontrols could be considered as population controls, this conditionalodds ratio is the appropriate estimate of the relative risk of rs2200733T versus the wild-type. Conditional odd-ratio for rs13143308 T issimilarly defined and has a similar interpretation.

Correction for Relatedness and Genomic Control.

Some of the individuals in the Icelandic case-control groups wererelated to each other, causing the aforementioned chi-square teststatistic to have a mean>1 and median>0.675²⁶. We estimated theinflation factor by using a previously described procedure where wesimulated genotypes through the genealogy of 731,175 Icelanders³⁰. Forthe initial discovery samples, where genotypes for the 316,515genome-wide scan SNPs were available, we also estimated the inflationfactor by using genomic controls and calculating the average of the316,515 chi-square statistics, and by computing the median of the316,515 chi-square statistics and dividing it by 0.675²⁶ as describepreviously^(31,32). For these initial samples the inflation factors,estimated by our genealogy method and the two genomic control methodsgave similar inflation factor estimates; 1.047, 1.058 and 1.054respectively. The P values and confidence intervals presented are basedon adjusting by the inflation factor estimated by the genealogy method.

PCR Screening of cDNA Libraries.To confirm the expression of the spliced ESTs (DA725631, DB324364 andAF017091) within the LD block we screened commercially available cDNAlibraries and libraries generated at deCODE. The commercial librariesscreened were heart (Clontech-639304), aorta (Clontech-639325) bonemarrow (Clontech 7416-1), testis (Clontech 7414-1) and whole brain (BD50598) Marathon Ready cDNA libraries. In addition cDNA libraries wereconstructed for whole blood and EBV-transformed human lymphoblastoidcells. Total RNA was isolated from the lymphoblastoid cell lines andwhole blood, using the RNeasy RNA isolation kit from Qiagen (Cat. 75144)and the RNeasy RNA isolation from whole blood kit (Cat. 52304),respectively. cDNA libraries were prepared at deCODE using High CapacitycDNA Archive Kit with random primers (Applied Biosystems PN 4322171).PCR screening was carried out using the Advantage® 2 PCR Enzyme RT_PCRSystem (Clontech) according to manufacturers instructions and using PCRprimers from Operon Biotechnologies. The PCR reactions were done in 10μl volume at a final concentration of 3.5 μM of forward and reverseprimers (Table 16), 2 mM dNTP, 1× Advantage 2 PCR buffer and 0.5 μl ofcDNA library.

Northern Blot Analysis.

Commercial multiple tissue poly-A Northern blots were obtained fromClontech (Human Cardiovascular system, Cat. 636825).

Probes Used:

i) The PITX2 cDNA clone (HU3_p983E0327D), obtained from RZPD DeutschesRessourcenzentrum für Genomforschung GmbH, Germanyhttp://www.rzpd.de/products/genomecube.shtml) (sequence verified, datanot shown);ii) cDNA clone that corresponded to exons 1-12 of the ENPEP transcriptsobtained from RT-PCR experiments. The ENPEP clone was sequence verified:

TCCTGCTCCAGCTTGTGGATATTTTGCAAAAAAGCTCTCCATCTGCCACAGTTGCAGTTCAGTGTTGAATGGCTCTGCTATTGTGACAATTCGGCCAAGGTTTCTGTTATTGAGTGTATATCTGTTGACTAGATAGTCCCAGTTGAGTTGTATCCAATTCCAGGCCATGTTCTTCCCATAGCTGTTATATGAGATATATCGAATGACTGTAAACACATCCTGAGTTTTAATAAGGTTCGTGTCCTTGAGCAAATCCAAATACCTTGACAAAAGAGTAACGTTCTTCACTGATGCTAATCCATACAGCAGTTTTTCTTTTTCTTGAGCTAATGAAGTTTTCTGGTATTGCTCAAGAGTGTAGTTCCATGAAATCTCATTGCCAGAGTTCTGCATCCCATACCGATACACCAGAAGCCTGAGATTTACGGGAAGGCTTACAGTCCCATTTAGCCACTGCTCAAATAACGAGGAAGCATTGTTCAAGGCTTCTCTGTCTCCCATCTTGCACGCAAACCCTAACACGGAGGAACGGAGTAACTTTGTGACATGGTCTCCAGCATCATTCCATCCCAGAGAATCTGCAATAGGCTTCACTTGACCTTGGAAGTATTCCTCAATCATAGGATATAGCTCTTTATCATCTTCAAACATGCTAATGATGTAGGTTACAGCTGAAATTACTCTCTGCCATGGTAAAAAATTCTCTTCCCTTTTGAGATACTTGGTCAAGTTCAAAGCCACCTTATAATCTAGAAGTTGAGCTCTTGCCAAGGCAAAAGCATCATCAATAAGACTTGCACGATCTGCTGAAGAAAATGTCTTGTGGTTCAAGGAGAGCGCTGTAGCTATCGAGTCCCAAGTTGCTACTTCATAATTTACACGATAAAACCCAATATGATCTGGGTTTATTTTGAGAAAAGCATTTCCACTAGGATTAGAGGAGTTCAAAGTGATTCCTTCTTTTTCTGACCTATTAAATAACACACTGCTTGTTATATTATCTTCAGTCCATTTAACTGGGATATTCCATGTATAACCAAGATCTGAAGGGGGCTGAGAAGGGTTAGCTCTTGGGTCCAACAAAAAGCGTTTCTGTGTGATGTTCTTGACACCGTTCACGTTAAGCACAGGATAACCCATCTgGTCTGGTCCAGGTGTCCATTACTTCTTTCACTGGTAGCCTACTTGCCTCTTCCAGTGCTGCCCAAAAATcDNA fragments were radiolabelled with [α-³²P]dCTP (specificactivity6000 Ci/mmol), using the Megaprime labeling kit (GE HealthcareCat. RPN 1607) and unincorporated nucleotides removed from the reactionusing ProbeQuant G-50 microcolumns (GE Healthcare Cat. 27-5335-01).Membranes were pre-hybridized in Rapid-hyb buffer (GE Healthcare Cat.RPN 1635) for at least 30 minutes and subsequently hybridized with100-300 ng of the labelled cDNA probe. Hybridizations were performed inRapid-hyb buffer at 65° C. overnight. The labelled probes were heatedfor 5 minutes at 95° C. before addition to the filters in thepre-hybridization solution. After hybridization, the membranes werewashed at low stringency in 2×SSC, 0.05% SDS at room temperature for30-40 minutes followed by two high stringency washes in 0.1×SSC, 0.1%SDS at 50° C. for 40 minutes. The blots were immediately sealed andexposed to Kodak BioMax MR X-ray film (Cat. 8715187).

Surveying for Candidate Regulatory Variants in the AF Region

The UCSC browser was used to extracted positions of SNPs and conservedtranscription factor binding sites (TFBS) for a 172.5 kb region aroundthe SNPs associated with AF (hg release 17, chromosome 4,111,942,401-112,114,901). The two tables were cross referenced and SNPsthat landed in binding sites were further interrogated for LD withrs2220427 or rs6843082 in the HapMap data. This was done for releases16, 17 and 18 of the human genome, but the results are reported in hg 17coordinates. This yielded 3 SNPs that land in conserved binding sitesfor known transcription factors (Table 17). Note, this analysis onlydetects a limited sample of functional candidates as i) the AFhaplotypes have not been sequenced fully, ii) several candidate SNPs arenot typed in Hapmap and it is unknown whether they sit on the AFhaplotypes, iii) polymorphisms in less conserved regions could befunctional.

Evolutionary Conservation of Three TFBS

Utilizing the Multiz alignment in the UCSC genome enabled an assessmentof the evolutionary conservation of the regions affected by these SNPs.In all three cases is the core part of the TF binding sites intact, butthe positions affected are preserved to a different degree. The SOX5affected by rs12510087 is least conserved in mammals but the second one(affected by rs2220427) is strikingly preserved (with the exception ofOpossum it is maintained in all species to the chicken). The rs17042171mutation is in last position in the core GGAAAA motif of the NFATbinding site. The conservation indicates that a G is preferred at thislocation, resulting in a GGAAAG motif.

Correlation Between Genotype and Expression of ENPEP

Blood was collected in the morning, between 8 and 10 am, after overnightfasting (from 9 pm) and RNA extracted within 2 hours from phlebotomyfrom 1,002 individuals. RNA isolation was performed using the RNeasyMidi Kit (QIAGEN GmbH, Hilden, Germany). Subcutaneous fat samples (5-10cm³) were removed through a 3 cm incision at the bikini line (alwaysfrom the same site to avoid site-specific variation) after localanesthesia using 10 ml of lidocaine-adrenalin (1%) from 673 individuals.Purification of the total RNA was performed with the RNeasy Mini Kit(QIAGEN GmbH, Hilden, Germany).

Integrity of the total RNA was assessed through analysis on the Agilent2100 Bioanalyzer (Agilent Technologies, Palo Alto, U.S., CA). Eachlabelled RNA sample including reference pools, 1,765 samples in total,was hybridized to a Human 25K array manufactured by AgilentTechnologies. Array images were processed as described previously toobtain background noise, single-channel intensity and associatedmeasurement error estimates¹¹. Expression changes between two sampleswere quantified as mean logarithm (log₁₀) expression ratio (MLR), i.e.expression ratios compared to background corrected intensity values forthe two channels for each spot on the arrayl². The hybridizations wentthrough standard QC process, i.e. signal to noise ratio, reproducibilityand accuracy at spike-in compounds, comparing Cy3 to Cy5 intensities.

Neither associated SNP was correlated to the expression of ENPEPadjusted for age and sex in blood (P=0.90 and P=0.82 for rs2200733 andrs10033464, respectively) or adipose tissue (P=0.23 and P=0.37 forrs2200733 and rs10033464, respectively)

TABLE 7 Analysis of the association of rs2200733 and rs10033464 onchromosome 4q25 to AF/AFl. Sample rs2200733 T^(a) rs10033464 T^(a, b) (Ncases/ OR OR Compa- Joint N controls) Freq.^(c) (95% CI) P Freq.^(c)(95% CI) P rison P^(d) PAR Iceland^(e) Discovery 0.191 1.84 2.0 × 10⁻¹¹0.110 1.42 0.0024 0.041  0.216 (550/4,476) 0.114 (1.54-2.21) 0.080(1.13-1.77) Replication 0.166 1.64 2.7 × 10⁻²³ 0.108 1.40 8.2 × 10⁻⁸ 0.028  0.176 (2,251/13,238) 0.108 (1.49-1.81) 0.080 (1.24-1.58) Combined0.171 1.68 1.9 × 10⁻³⁰ 0.108 1.40 9.4 × 10⁻⁹  0.0025  0.180(2,801/17,714) 0.110 (1.53-1.83) 0.080 (1.25-1.55) Other Europeanancestry Sweden 0.179 2.01 0.00027 0.172 1.65 0.0087 0.41   0.272(143/738) 0.098 (1.38-2.93) 0.111 (1.14-2.41) U.S. 0.229 1.84 9.8 ×10⁻¹⁰ 0.105 1.30 0.052  0.026  0.232 (636/804) 0.139 (1.51-2.23) 0.083(1.00-1.69) Combined^(f) — 1.88 1.2 × 10⁻¹² — 1.41 0.0019 0.027  0.237 —(1.58-2.23) — (1.13-1.75) All European ancestry Combined^(f) — 1.72 3.3× 10⁻⁴¹ — 1.39 6.9 × 10⁻¹¹ 0.00019 0.206 — (1.59-1.86) — (1.26-1.53)Hong Kong Hong Kong 0.605 1.42 0.00064 0.190 1.08 0.55  0.0099  0.346(333/2,836) 0.528 (1.16-1.73) 0.218 (0.84-1.39) Each row contains theresults from a joint analysis of two variants, rs2200733 T andrs10033464 T^(b). The numbers of cases and controls (N) are shown foreach case-control study and for each variant the allelic frequencies ofthe variant in cases and controls, the OR with a 95% CI and two- sided Pvalues, are shown. In addition a P value for comparing the effect of thetwo variants and their joint population attributable risk (PAR) isreported. For example, the first row indicates that, for the initialIcelandic discovery samples, rs2200733 T has an estimated odds ratio(OR) of 1.84 (95% CI (1.54-2.21), P = 4.1 × 10⁻¹¹) vs the wild type(rs2200733 C, rs13143308 G, rs10033464 G haplotype), and rs10033464 Thas an estimated OR of 1.42 (95% CI (1.13-1.77), P = 0.0024) vs the wildtype. ^(a)Results of comparing rs2200733 T and rs10033464 T to the wildtype rs2200733 C, rs13143308 G, rs10033464 G haplotype. ^(b)In theSwedish and the U.S. samples rs10033464 T was tagged by the rs2200733 C,rs13143308 T haplotype ^(c)The frequency in cases (above) and controls(below) ^(d)P value for comparing the ORs of rs2200733 T and rs10033464T. ^(e)The association analysis was adjusted for the relatedness of someof the individuals. ^(f)For the combined study populations of Europeandecent, the PAR was calculated by using the average, unweighted controlfrequency of the populations, while the OR and the P value wereestimated using the Mantel-Haenszel model.

TABLE 8 Association by age at diagnosis in Iceland and by AFsub-phenotype in the U.S. Sample rs2200733^(a) rs10033464^(a, b) (Ncases/ Male OR OR N controls) % Age ± SD (95% CI) (95% CI) P Sex PIceland^(c) Diagn. ≦60 77.8 50.7 ± 8.4  2.12 1.69 6.3 × 10⁻¹⁸ 0.82(510/17,714) (1.77-2.54) (1.34-2.12) Diagn. 60-70 66.2 65.6 ± 2.9  1.881.44 6.7 × 10⁻¹⁵ 0.58 (654/17,714) (1.60-2.21) (1.18-1.77) Diagn. 70-8058.9 75. 0 ± 2.8  1.60 1.23 7.5 × 10⁻¹¹ 0.96 (958/17,714) (1.39-1.84)(1.03-1.47) Diagn. >80 47.4 85.6 ± 4.2  1.20 1.31 0.0044 0.36(679/17,714) (1.01-1.43) (1.08-1.60) U.S. Lone AF 81.7 46.1 ± 11.5 2.321.68 1.2 × 10⁻¹⁰ 0.46 (251/804) (1.80-2.99) (1.19-2.37) AF/HTN 74.6 54.5± 10.2 2.23 1.66 0.0010 0.54 (67/804) (1.43-3.48) (0.90-3.04) Other AF52.8 75.2 ± 11.3 1.44 0.97 0.015 0.85 (318/804) (1.12-1.84) (0.69-1.37)Each row contains the results from a joint analysis of two variants,rs2200733 T and rs10033464 T^(a). The numbers of cases and controls (N),the percentage of male cases, and the mean age (±SD) for cases, areshown for each case-control study. The OR, with a 95% CI, and P valuesare shown for each variant. In addition a joint P value for the combinedeffect of the two variants, and a joint P value for testing if there isa difference of the allelic frequency of the variants between the sexeswithin each sub-group of patients. ^(a)Results of comparing rs2200733 Tand rs10033464 T to the wild type rs2200733 C, rs13143308 G, rs10033464G haplotype. ^(b)In the U.S. samples rs10033464 T was tagged by thers2200733 C, rs13143308 T haplotype. ^(c)The association analysis wasadjusted for the relatedness of some of the individuals.

TABLE 9 SNPs equivalent to rs10033464, rs13143308 and rs2200733 in CEUHapMap data Tagging SNP SNP Build 35 location SEQ ID NO: 50 locationrs12503217 rs10033464 112063765 108955 rs12510087 rs10033464 112066632111822 rs6852357 rs10033464 112071939 117129 rs4400058 rs10033464112074277 119467 rs10033464 rs10033464 112078365 123555 rs2171592rs10033464 112078392 123582 rs2350539 rs10033464 112078814 124004rs1906606 rs10033464 112080996 126186 rs723364 rs10033464 112082075127265 rs2220429 rs10033464 112085089 130279 rs4032976 rs10033464112086371 131561 rs3853440 rs10033464 112087213 132403 rs3853441rs10033464 112087344 132534 rs3853442 rs10033464 112087632 132822rs3853443 rs10033464 112087733 132923 rs4124158 rs10033464 112087798132988 rs4124159 rs10033464 112087847 133037 rs12506083 rs10033464112088016 133206 rs4032975 rs10033464 112089842 135032 rs4032974rs10033464 112090140 135330 rs2634074 rs13143308 112034645 79835rs2466455 rs13143308 112043219 88409 rs2723334 rs13143308 11204635691546 rs1906616 rs13143308 112055172 100362 rs1906615 rs13143308112059402 104592 rs2129983 rs13143308 112061684 106874 rs2129982rs13143308 112061747 106937 rs1906599 rs13143308 112070290 115480rs13143308 rs13143308 112072023 117213 rs6843082 rs13143308 112075671120861 rs17042059 rs2200733 111998790 43980 rs4529121 rs2200733112003159 48349 rs4543199 rs2200733 112005744 50934 rs12647316 rs2200733112006855 52045 rs10019689 rs2200733 112007473 52663 rs4626276 rs2200733112007593 52783 rs17042076 rs2200733 112009942 55132 rs11098089rs2200733 112011830 57020 rs17042088 rs2200733 112012418 57608rs11930528 rs2200733 112017798 62988 rs17042098 rs2200733 11202176266952 rs17042102 rs2200733 112026230 71420 rs17042121 rs2200733112034705 79895 rs10516563 rs2200733 112035326 80516 rs4605724 rs2200733112042685 87875 rs2350269 rs2200733 112044728 89918 rs6533527 rs2200733112045118 90308 rs17042144 rs2200733 112047270 92460 rs1906618 rs2200733112053026 98216 rs1906617 rs2200733 112053418 98608 rs12646447 rs2200733112056930 102120 rs12646754 rs2200733 112061176 106366 rs2129981rs2200733 112061803 106993 rs12639654 rs2200733 112062899 108089rs6817105 rs2200733 112063372 108562 rs17042171 rs2200733 112065891111081 rs1906591 rs2200733 112066493 111683 rs1906592 rs2200733112066608 111798 rs2200732 rs2200733 112067646 112836 rs2200733rs2200733 112067773 112963 rs4611994 rs2200733 112068645 113835rs4540107 rs2200733 112068706 113896 rs1906593 rs2200733 112069526114716 rs1906596 rs2200733 112069840 115030 rs2220427 rs2200733112072493 117683

TABLE 10 Haplotype structure (haplotypes with estimated frequency >0.1%)over key SNPs and the D4S406 microsatellite in Iceland Frequency D4S406rs2200733 rs13143308 rs10033464 0.0800 −8 T T G 0.00647 −6 C T T 0.00225−4 T T G 0.0415 −2 T T G 0.00108 0 T T G 0.0592 0 C T T 0.00679 2 C T T0.0169 2 C G G 0.00923 4 C T T 0.135 4 C G G 0.0853 6 C G G 0.1587 8 C GG 0.163 10 C G G 0.0928 12 C G G 0.0398 14 C G G 0.101 16 C G G

TABLE 11 Association to all Hap300 IIlumina SNPs in a 200 kb regionaround rs2200733 and rs10033464 in an extended set of Icelandic AF/AFlcases and controls. Results have not been adjusted for relatedness ofindividuals. Adjusting for Also adjusting rs2220427 for rs10033464 SNPLocation All. Freq OR P value OR P value OR P value rs4834295 111892810G 0.817 1.0 0.27 1.0 0.39 1.03 0.63 rs2278782 111899758 C 0.883 1.0 0.790.9 0.70 0.99 0.93 rs2595110 111902927 T 0.637 1.0 0.13 1.0 1.0 1.010.89 rs976568 111908325 A 0.743 1.0 0.83 0.9 0.62 0.97 0.58 rs2197815111924481 T 0.030 1.1 0.34 1.1 0.34 0.97 0.84 rs2723286 111940938 A0.231 1.0 0.26 1.0 0.50 1.03 0.59 rs2723296 111962087 G 0.229 1.0 0.381.0 0.60 1.03 0.67 rs1699716 111986643 T 0.153 1.3 4.7 × 10⁻⁵ 0.9 0.590.95 0.53 rs2723316 111991891 T 0.297 1.2 1.9 × 10⁻⁵ 1.0 0.59 0.95 0.40rs6419178 111993104 A 0.143 1.1 0.17 1.0 0.25 0.98 0.77 rs1448817111998657 G 0.252 1.4 4.2 × 10⁻  1.1 0.035 1.06 0.46 rs2634073 112023387A 0.167 1.6 2.4 × 10⁻  1.2 0.039 0.90 0.48 rs2200733 112067773 T 0.1191.7 7.6 × 10⁻  — — — — rs2220427 112072493 T 0.120 1.7 5.6 × 10⁻  — — —— rs1310587 112075751 C 0.888 1.0 0.33 0.9 0.89 0.95 0.56 rs1003346112078365 T 0.082 1.2  0.013 1.3 5.1 × 10⁻⁴ — — rs1314119 112086218 A0.368 1.3 2.0 × 10⁻  1.1 0.0067 1.08 0.29 rs3853444 112091740 A 0.6041.1  0.053 1.0 0.45 1.06 0.24

TABLE 12 Association study of SNPs which are equivalent to rs2200733 inCEU HapMap samples in the Chinese samples from Hong Kong. SNP LocationAll. Freq OR P value HapMap D′ HapMap R² rs11930528 112017798 T 0.4721.27 0.011  0.91 0.66 rs17042121 112034705 G 0.418 1.32 0.0029 0.97 0.64rs6533527 112045118 A 0.518 1.37 0.0014 0.95 0.79 rs1906617 112053418 C0.524 1.35 0.0026 1.00 0.98 rs12639654 112062899 T 0.519 1.39 0.00121.00 1.00 rs2200733 112067773 T 0.516 1.42 6.4 × 10⁻⁴ — — rs4611994112068645 C 0.518 1.39 0.0012 1.00 1.00 The LD values reported are tors2200733 in the combined CHB and JPT HapMap samples

TABLE 13 Association to AF/AFl by genotype Allelic RR Genotype RR 1 2 0001 02 11 12 22 P value Iceland 1.68 1.38 1 1.55 1.36 3.42 2.47 1.58 0.12Sweden 2.01 1.65 1 1.66 1.72 5.86 3.10 2.04 0.68 U.S. 1.84 1.30 1 1.631.40 4.86 2.31 0.90 0.25 Combined 1.71 1.38 1 1.56 1.37 3.64 2.44 1.430.018 Hong Kong 1.42 1.07 1 1.15 0.95 1.77 1.34 0.97 0.87 The threepossible haplotypes are coded as 0 = rs2200733 C, rs13143308 G,rs10033464 G 1 = rs2200733 T, rs13143308 T, rs10033464 G 2 = rs2200733C, rs13143308 T, rs10033464 T

TABLE 14 Association of various phenotypes, considered risk factors forAF to risk variants. Phenotype T rs2200733 T rs10033464 (N cases/Ncontrols) OR P value OR P value Hypertension 1.08 0.11 1.05 0.37(2,620/19,862) Myocardial infarction 1.05 0.26 1.04 0.49 (3,576/19,542)Obesity-BMI >35 0.96 0.51 1.00 1.00 (1,601/21,593)

TABLE 15 A summary of the source of the Icelandic controls. Note thatindividuals may come from multiple project and that some individuals mayhave been collected as relatives of probands. Frequency of Frequency ofSource Project Count T rs2200733 T rs10033464 Discovery ControlsAddiction 376 0.096 0.082 Anxiety 337 0.110 0.088 Breast Cancer 8760.116 0.085 Colon Cancer 370 0.119 0.070 Infectious Disease 297 0.1090.096 MI 454 0.104 0.076 Population Controls 389 0.099 0.077 ProstateCancer 713 0.123 0.081 Schizophrenia 291 0.110 0.091 Type II Diabetes551 0.102 0.078 Replication Controls Breast Cancer 228 0.122 0.074 TypeII Diabetes 340 0.097 0.082 Alzheimer 459 0.107 0.061 Osteoarthritis1,175 0.107 0.081 PAD 479 0.096 0.083 COPD 326 0.125 0.082 Stroke 4140.092 0.069 Osteoporosis 1,155 0.109 0.072 MI 390 0.112 0.075Hypertension 210 0.118 0.101 Depression 152 0.128 0.061 Asthma 538 0.1060.076 Parkinson 173 0.102 0.058 Population Controls 305 0.105 0.097Ankylosing Spondylitis 155 0.095 0.077 Sleep Apnea 422 0.118 0.074 AMD442 0.101 0.067 Rheumatoid Arthritis 430 0.100 0.094 Lung Cancer 2370.106 0.084 FCH 265 0.112 0.057 Longevity 392 0.09 0.077 BenignProstatic Hyperplasia 245 0.101 0.058 Pre-eclampsia 262 0.129 0.083Enuresis 249 0.104 0.087 Migrane 590 0.112 0.085 Myopia 353 0.123 0.085Thyroid Cancer 104 0.121 0.097 ADHD 123 0.119 0.089 Prostate Cancer 5800.117 0.073 Anxiety 546 0.121 0.096 Obesity 162 0.081 0.092Endometriosis 258 0.106 0.084 Kidney Cancer 174 0.099 0.100 Melanoma 2830.088 0.089 Addiction 201 0.138 0.098 Psoriasis 392 0.136 0.079 IBD 3560.093 0.102

TABLE 16 Primers used for ESTs screening of cDNA libraries ESTs*Forward primer Reverse primer DA725631 AGTGGAGGCTGCCAGACTTCTGCACCACTCATCACCAACA DB324364 CCGAGGATGTCTTTAGTCTGCAAATCATACAGCAGGAATGCAAACA AF017091 TGAGATTCCACATCCAACATCTTTTGGCAAACTTGATATTGTTCTTG *EST names are from NCBI BUILD 35

TABLE 17 SNPs that land in conserved TFBS in the region associated withAF. SNP Location Strand Ancestral Polym. TFBS TF start TF end rs17042171112065890 + C NC NFAT 112065889 112065900 rs12510087 112066631 + A NGSOX5 112066632 112066641 rs2220427 112072492 + C C/T SOX5 112072483112072493 Strand indicates the strand in genome alignment that themutation lands in. Polym. is the two alleles of the polymorphism at thissite.

TABLE 18 Markers in or near the PITX2 gene in LD with markers in the LDblock C04. Shown are markers in or near PITX2 (marker 1) and theircorrelation to markers in LD block C04 (marker 2). Marker 1 Marker 2 D′r2 p-value rs7668322 rs10033464 0.46291 0.133423 0.000953 rs2197815rs10033464 0.660377 0.300172 2.55E−06 rs6831623 rs2200733 1 0.028340.025473 rs2595110 rs2200733 0.699643 0.02996 0.067245

TABLE 19 Markers in linkage disequilibrium with marker rs2220427 andmarkerrs10033464 by values for r² of greater than 0.1. LD was calculatedbased on the HapMap CEU population sample. Pos in Pos in SEQ ID Marker 1anchor D′ r2 P-value B35 NO: 50 rs9994891 rs2220427 1 0.128329 0.002914111149057 rs11568995 rs2220427 1 0.128329 0.002914 111255189 rs4698804rs2220427 1 0.128329 0.002914 111297649 rs721413 rs2220427 1 0.1283290.002914 111305212 rs10488883 rs2220427 1 0.128329 0.002914 111305486rs6854883 rs2220427 0.788889 0.510189 4.17E−09 111964919 10109 rs2255793rs2220427 1 0.245283 9.27E−09 111965457 10647 rs2723298 rs2220427 10.274924 8.39E−09 111966089 11279 rs2723300 rs2220427 1 0.2365071.40E−08 111972512 17702 rs2723307 rs2220427 1 0.176558 2.98E−07111975800 20990 rs1584429 rs2220427 1 0.245283 9.27E−09 111976151 21341rs1448799 rs2220427 1 0.245283 9.27E−09 111980386 25576 rs1448798rs2220427 1 0.245283 9.27E−09 111980789 25979 rs1900827 rs2220427 10.246741 8.60E−08 111981343 26533 rs2197814 rs2220427 1 0.2405061.20E−08 111983098 28288 rs969642 rs2220427 1 0.245283 9.27E−09111983529 28719 rs2595093 rs2220427 0.830131 0.513828 1.59E−10 11198496030150 rs2245595 rs2220427 1 0.252078 6.90E−09 111985715 30905 rs2595088rs2220427 1 0.254302 1.99E−08 111985958 31148 rs981150 rs2220427 10.245283 9.27E−09 111986232 31422 rs16997168 rs2220427 0.819277 0.5074512.11E−10 111986643 31833 rs16997169 rs2220427 1 0.245283 9.27E−09111986685 31875 rs4527540 rs2220427 1 0.245283 9.27E−09 111986742 31932rs17042026 rs2220427 0.833488 0.554106 1.23E−11 111989978 35168rs2723316 rs2220427 1 0.245283 9.27E−09 111991891 37081 rs2595081rs2220427 0.832621 0.549261 3.07E−11 111992761 37951 rs2595085 rs22204271 0.242283 1.16E−08 111994377 39567 rs2723318 rs2220427 1 0.2365071.40E−08 111994576 39766 rs1448817 rs2220427 1 0.296277 9.75E−10111998657 43847 rs17042059 rs2220427 1 1 1.62E−20 111998790 43980rs4529121 rs2220427 1 1 1.43E−20 112003159 48349 rs10032150 rs2220427 10.296277 9.75E−10 112004222 49412 rs4543199 rs2220427 1 1 1.43E−20112005744 50934 rs12647316 rs2220427 1 1 1.43E−20 112006855 52045rs12647393 rs2220427 1 0.917379 1.57E−15 112006886 52076 rs10019689rs2220427 1 1 1.43E−20 112007473 52663 rs4626276 rs2220427 1 1 1.43E−20112007593 52783 rs17042076 rs2220427 1 1 1.62E−20 112009942 55132rs11098089 rs2220427 1 1 1.62E−20 112011830 57020 rs17042088 rs2220427 11 1.62E−20 112012418 57608 rs11944778 rs2220427 0.91509 0.8116424.20E−12 112014571 59761 rs4307025 rs2220427 1 0.296277 9.75E−10112015107 60297 rs11930528 rs2220427 1 1 1.42E−19 112017798 62988rs17042098 rs2220427 1 1 1.43E−20 112021762 66952 rs2634073 rs2220427 10.523052 1.42E−12 112023387 68577 rs17042102 rs2220427 1 1 2.07E−16112026230 71420 rs2634071 rs2220427 1 0.528302 2.16E−13 112026824 72014rs2634074 rs2220427 1 0.433962 5.12E−12 112034645 79835 rs17042121rs2220427 1 1 1.43E−20 112034705 79895 rs10516563 rs2220427 1 1 1.43E−20112035326 80516 rs4605724 rs2220427 1 1 1.43E−20 112042685 87875rs2466455 rs2220427 1 0.491956 5.72E−12 112043219 88409 rs2350269rs2220427 1 1 1.42E−19 112044728 89918 rs6533527 rs2220427 1 1 1.43E−20112045118 90308 rs2723334 rs2220427 1 0.43396 25.12E−12  112046356 91546rs17042144 rs2220427 1 1 1.43E−20 112047270 92460 rs1906618 rs2220427 11 2.67E−19 112053026 98216 rs1906617 rs2220427 1 1 1.43E−20 11205341898608 rs6847935 rs2220427 1 0.921053 1.10E−18 112054255 99445 rs1906616rs2220427 1 0.433962 5.12E−12 112055172 100362 rs12646447 rs2220427 1 11.84E−20 112056930 102120 rs1906615 rs2220427 1 0.433962 5.12E−12112059402 104592 rs12646754 rs2220427 1 1 2.08E−20 112061176 106366rs2129983 rs2220427 1 0.428571 6.61E−12 112061684 106874 rs2129982rs2220427 1 0.433962 5.12E−12 112061747 106937 rs2129981 rs2220427 1 11.43E−20 112061803 106993 rs12639654 rs2220427 1 1 1.43E−20 112062899108089 rs6817105 rs2220427 1 1 1.62E−20 112063372 108562 rs17042171rs2220427 1 1 1.43E−20 112065891 111081 rs1906591 rs2220427 1 1 1.43E−20112066493 111683 rs1906592 rs2220427 1 1 1.26E−19 112066608 111798rs2200732 rs2220427 1 1 1.85E−19 112067646 112836 rs2200733 rs2220427 11 1.43E−20 112067773 112963 rs4611994 rs2220427 1 1 1.43E−20 112068645113835 rs4540107 rs2220427 1 1 1.43E−20 112068706 113896 rs1906593rs2220427 1 1 1.62E−20 112069526 114716 rs1906596 rs2220427 1 1 2.68E−20112069840 115030 rs1906599 rs2220427 1 0.433962 5.12E−12 112070290115480 rs13143308 rs2220427 1 0.438445 5.36E−12 112072023 117213rs6843082 rs2220427 1 0.433962 5.12E−12 112075671 120861 rs11931959rs2220427 1 0.249653 7.85E−09 112077289 122479 rs13121924 rs2220427 10.156089 9.36E−07 112078423 123613 rs2129979 rs2220427 1 0.2567895.78E−09 112078601 123791 rs723363 rs2220427 1 0.156089 9.36E−07112082105 127295 rs7697491 rs2220427 1 0.154058 1.07E−06 112083422128612 rs13141190 rs2220427 1 0.156089 9.36E−07 112086218 131408rs6533530 rs2220427 1 0.156089 9.36E−07 112089540 134730 rs6533531rs2220427 1 0.156089 9.36E−07 112089569 134759 rs3866831 rs2220427 10.156089 9.36E−07 112089718 134908 rs3866832 rs2220427 0.857992 0.1096030.000186 112091304 136494 rs11098083 rs10033464 0.407276 0.12964 0.00205111855920 rs11721423 rs10033464 0.365905 0.11129 0.003321 111858873rs10005945 rs10033464 0.433962 0.101848 0.004763 111860013 rs7668322rs10033464 0.46291 0.133423 0.000953 111906200 rs2197815 rs100334640.660377 0.300172 2.55E−06 111924481 rs6831623 rs10033464 1 0.5112363.33E−10 111964677 9867 rs7661383 rs10033464 0.637611 0.21478 0.000011111979181 24371 rs7667461 rs10033464 0.637611 0.21478 0.000011 11197973824928 rs1900827 rs10033464 0.735008 0.134278 0.000368 111981343 26533rs998101 rs10033464 0.635887 0.20914 0.000014 111988219 33409 rs12646859rs10033464 0.719793 0.271646 0.000191 111992237 37427 rs12498380rs10033464 0.60232 0.178165 0.000097 111992563 37753 rs7690164rs10033464 0.496308 0.148782 0.005455 111994069 39259 rs11098090rs10033464 0.551083 0.169161 0.000104 112014012 59202 rs2634073rs10033464 0.640189 0.223694 8.35E−06 112023387 68577 rs2634071rs10033464 0.637611 0.21478 0.000011 112026824 72014 rs2634074rs10033464 1 0.433962 5.12E−12 112034645 79835 rs2466455 rs10033464 10.428256 1.77E−09 112043219 88409 rs2723334 rs10033464 1 0.4339625.12E−12 112046356 91546 rs1906616 rs10033464 1 0.433962 5.12E−12112055172 100362 rs1906615 rs10033464 1 0.433962 5.12E−12 112059402104592 rs2129983 rs10033464 1 0.428571 6.61E−12 112061684 106874rs2129982 rs10033464 1 0.433962 5.12E−12 112061747 106937 rs12503217rs10033464 1 1 1.43E−20 112063765 108955 rs12510087 rs10033464 1 11.43E−20 112066632 111822 rs1906599 rs10033464 1 0.433962 5.12E−12112070290 115480 rs6852357 rs10033464 1 1 1.43E−20 112071939 117129rs13143308 rs10033464 1 0.421583 2.22E−11 112072023 117213 rs4833456rs10033464 1 0.923858 6.67E−19 112073911 119101 rs4400058 rs10033464 1 11.43E−20 112074277 119467 rs6843082 rs10033464 1 0.433962 5.12E−12112075671 120861 rs2171592 rs10033464 1 1 1.43E−20 112078392 123582rs13121924 rs10033464 1 0.156089 9.36E−07 112078423 123613 rs2350539rs10033464 1 1 1.43E−20 112078814 124004 rs1906606 rs10033464 1 11.43E−20 112080996 126186 rs723364 rs10033464 1 1 1.43E−20 112082075127265 rs723363 rs10033464 1 0.156089 9.36E−07 112082105 127295rs7697491 rs10033464 1 0.154058 1.07E−06 112083422 128612 rs2220429rs10033464 1 1 1.43E−20 112085089 130279 rs13141190 rs10033464 10.156089 9.36E−07 112086218 131408 rs4032976 rs10033464 1 1 1.62E−20112086371 131561 rs3853440 rs10033464 1 1 1.62E−20 112087213 132403rs3853441 rs10033464 1 1 1.43E−20 112087344 132534 rs3853442 rs100334641 1 1.43E−20 112087632 132822 rs3853443 rs10033464 1 1 1.43E−20112087733 132923 rs4124158 rs10033464 1 1 1.10E−17 112087798 132988rs4124159 rs10033464 1 1 1.43E−20 112087847 133037 rs12506083 rs100334641 1 1.43E−20 112088016 133206 rs6533530 rs10033464 1 0.156089 9.36E−07112089540 134730 rs6533531 rs10033464 1 0.156089 9.36E−07 112089569134759 rs3866831 rs10033464 1 0.156089 9.36E−07 112089718 134908rs4032975 rs10033464 1 1 7.06E−20 112089842 135032 rs4032974 rs100334641 1 1.43E−20 112090140 135330 rs3866832 rs10033464 1 0.148936 1.44E−06112091304 136494 rs7654080 rs10033464 1 0.151515 0.002495 112585323

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Science 299, 251-4 (2003).-   7. Yang, Y. et al. Identification of a KCNE2 gain-of-function    mutation in patients with familial atrial fibrillation. Am J Hum    Genet 75, 899-905 (2004).-   8. Xla, M. et al. A Kir2.1 gain-of-function mutation underlies    familial atrial fibrillation. Biochem Biophys Res Commun 332, 1012-9    (2005).-   9. Olson, T. M. et al. Kv1.5 channelopathy due to KCNA5    loss-of-function mutation causes human atrial fibrillation. Hum Mol    Genet 15, 2185-91 (2006).-   10. Hong, K., Bjerregaard, P., Gussak, I. & Brugada, R. Short QT    syndrome and atrial fibrillation caused by mutation in KCNH2. J    Cardiovasc Electrophysiol 16, 394-6 (2005).-   11. Ellinor, P. T. et al. Mutations in the long QT gene, KCNQ1, are    an uncommon cause of atrial fibrillation. Heart 90, 1487-8 (2004).-   12. Ellinor, P. T., Petrov-Kondratov, V. I., Zakharova, E.,    Nam, E. G. & MacRae, C. A. Potassium channel gene mutations rarely    cause atrial fibrillation. BMC Med Genet 7, 70 (2006).-   13. Franco, D. & Campione, M. The role of Pitx2 during cardiac    development. Linking left-right signaling and congenital heart    atrial fibrillation and/or strokes. Trends Cardiovasc Med 13, 157-63    (2003).-   14. Faucourt, M., Houliston, E., Besnardeau, L., Kimelman, D. &    Lepage, T. The pitx2 homeobox protein is required early for endoderm    formation and nodal signaling. Dev Biol 229, 287-306 (2001).-   15. Mommersteeg, M. T. et al. Molecular Pathway for the Localized    Formation of the Sinoatrial Node. Circ Res (2007).-   16. A haplotype map of the human genome. Nature 437, 1299-320    (2005).-   17. Waldo, A. L. The interrelationship between atrial fibrillation    and atrial flutter. Prog Cardiovasc Dis 48, 41-56 (2005).-   18. Zini, S. et al. Identification of metabolic pathways of brain    angiotensin II and III using specific aminopeptidase inhibitors:    predominant role of angiotensin III in the control of vasopressin    release. Proc Natl Acad Sci USA 93, 11968-73 (1996).-   19. Gretarsdottir, S. et al. The gene encoding phosphodiesterase 4D    confers risk of ischemic stroke. Nat Genet 35, 131-8 (2003).-   20. Falk, C. T. & Rubinstein, P. Haplotype relative risks: an easy    reliable way to construct a proper control sample for risk    calculations. Ann Hum Genet 51 (Pt 3), 227-33 (1987).-   21. Mantel, N. & Haenszel, W. Statistical aspects of the analysis of    data from retrospective studies of atrial fibrillation and/or    stroke. J Natl Cancer Inst. 22, 719-48 (1959).-   22. Grant, S. F. et al. Variant of transcription factor 7-like 2    (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38, 320-3    (2006).-   23. Yang, X. et al. Development and validation of stroke risk    equation for Hong Kong Chinese patients with type 2 diabetes: the    Hong Kong Diabetes Registry. Diabetes Care 30, 65-70 (2007).-   24. Baum, L. et al. Methylenetetrahydrofolate reductase gene A222V    polymorphism and risk of ischemic stroke. Clin Chem Lab Med 42,    1370-6 (2004).-   25. Kutyavin, I. V. et al. A novel endonuclease IV post-PCR    genotyping system. Nucleic Acids Research 34, e128 (2006).-   26. Amundadottir, L. T. et al. A common variant associated with    prostate cancer in European and African populations. Nat Genet 38,    652-8 (2006).-   27. Gretarsdottir, S. et al. The gene encoding phosphodiesterase 4D    confers risk of ischemic stroke. Nat Genet 35, 131-8 (2003).-   28. Falk, C. T. & Rubinstein, P. Haplotype relative risks: an easy    reliable way to construct a proper control sample for risk    calculations. Ann Hum Genet 51 (Pt 3), 227-33 (1987).-   29. Mantel, N. & Haenszel, W. Statistical aspects of the analysis of    data from retrospective studies of atrial fibrillation and/or    stroke. J Natl Cancer Inst. 22, 719-48 (1959).-   30. Grant, S. F. et al. Variant of transcription factor 7-like 2    (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38, 320-3    (2006).-   31. Devlin, B. & Roeder, K. Genomic Control for association studies.    Biometrics 55, 997-1004 (1999).-   32. Devlin, B., Bacanu, S.-A. & Roeder, K. Genomic control to the    extreme. Nature Genetics 36, 1129-1130 (2004).-   33. Nomenclature and criteria for diagnosis of ischemic heart atrial    fibrillation and/or stroke. Report of the Joint International    Society and Federation of Cardiology/World Health Organization task    force on standardization of clinical nomenclature. Circulation 59,    607-9 (1979).-   34. Alpert, J. S., Thygesen, K., Antman, E. & Bassand, J. P.    Myocardial infarction redefined—a consensus document of The Joint    European Society of Cardiology/American College of Cardiology    Committee for the redefinition of myocardial infarction. J Am Coil    Cardiol 36, 959-69 (2000).-   35. Monks, S. A. et al. Genetic inheritance of gene expression in    human cell lines. Am J Hum Genet 75, 1094-105 (2004).-   36. Schadt, E. E. et al. Genetics of gene expression surveyed in    maize, mouse and man. Nature 422, 297-302 (2003).

Example 3 Association of Chromosome 4 Variants to Ischemic Stroke

Stroke is a common cause of death and the leading cause of adultdisability in Western societies. It is now also becoming a major healthproblem in low-income and middle-income countries due to populationageing and changes in modifiable risk factors for cardiovasculardiseases¹. Stroke is not a single disease but a highly complex syndromeconsisting of a group of heterogeneous disorders with many genetic andenvironmental risk factors^(2,3). Studies on twins, family history andanimal models⁴⁻⁸ provide evidence for genetic contribution to the commonforms of stroke but no major risk variant has yet been identifiedshowing consistent results across populations.

Ischemic strokes (IS), accounting for the majority of cerebral insults(>80%), result from thrombosis or embolism leading to obstruction ofcerebral arteries. Various pathophysiological mechanisms can cause ISbut the most common ones are large artery atherosclerosis (LAA),cardioembolic stroke (CES) and small vessel disease (SVD)⁹.

Methods Study Populations.

Iceland:

Icelandic stroke patients were recruited from a registry of over 4,000individuals diagnosed with ischemic stroke or TIA at the only universityhospital in Reykjavik, the Landspitali University Hospital, during theyears 1993 to 2006. Stroke patients have been enrolled over the lastnine years through the cardiovascular disease (CVD) genetics program atdeCODE. Stroke diagnosis was clinically confirmed by neurologists (seebelow). The discovery cohort included 1,661 patients and when analysingthe SNPs on 4q25 we used an additional set of 282 patients (mean age±SD:77.2±11.3 years, 45% females). We used 25,708 controls (mean age±SD:59.2±21.1 years, 59% females) from various genetic programs under studyat Decode, including: abdominal aneurysm (250), atrial fibrillation(1,150), addiction (750), Alzheimer (350), anxiety (200), asthma (1300),COPD (850), colon cancer (200), deep vein thrombosis (550), dyslexia(200), infection diseases (250), longevity (400), lung cancer (750),myocardial infarction (2,400), migraine (1,100), peripheral arterydisease (1,200), polycystic ovary syndrome (1,200), pre-eclampsia (700),prostate cancer (400), psoriasis (750), rheumatic arthritis (550),restless leg syndrome (350), and type 2 diabetes (400).

The study was approved by the Data Protection Commission of Iceland(DPC) and the National Bioethics Committee of Iceland. All participantsgave informed consent.

Sweden:

Swedish patients with ischemic stroke attending the stroke unit or thestroke outpatient clinic at Karolinska University Hospital, Huddingeunit in Stockholm, Sweden, were recruited from 1996 to 2002 as part ofan ongoing genetic epidemiology study, the South Stockholm IschemicStroke Study (SSISS) (mean age±SD: 67.3±11.8 years, 44% females). TheSwedish controls used in this study are population-based controlsrecruited from the same region in central Sweden as the patients,representing the general population in this area. The individuals wereeither blood donors recruited at the Huddinge or Karolinska UniversityHospitals or healthy volunteers (recruited in 1990-1994) recruited bythe Clinical Chemistry Department at the Karolinska University Hospitalto represent a normal reference population (mean age±SD: 46.8115.9 yearsfor controls from Huddinge hospital, 41% females, age information notavailable for blood donors recruited at the Karolinska hospital). Thestudy was approved by the Bioethics Committee of Karolinska Institutet.

South-Germany:

The German population, herein referred to as Germany-S, consisted of ISpatients consecutively recruited during the period 2001-2006 at thestroke unit of the Department of Neurology, Klinikum Grosshadern,University of Munich, Germany (mean age±SD: 65.3±13.7 years, 38%females). The control group consisted of age and gender matchedindividuals without a history of cardiovascular disease (mean age±SD:62.7±10.9 years, 38% females). These were selected from the KORA S4study, a community based epidemiological project near Munich²³. Thestudy was approved by the local ethics committee and informed consentwas obtained from all individuals (or relatives or legal guardians).

Westphalia Region, Germany:

The second German population, referred to as Germany-W, recruitedischemic stroke patients through hospitals participating in the regionalWestphalian Stroke Register, located in the west of the country, duringthe period 2000-2003 (mean age±SD: 70.4±12.6 years, 53% females).Population controls without a self-reported history of stroke were drawnfrom the cross-sectional, prospective, population based Dortmund HealthStudy²⁴, conducted in the same region, and subsequently frequencymatched to the cases (mean age±SD: 52.3±13.7 years, 53% females). Bothstudies were approved by the ethics committee of the University ofMunster. All participants gave their informed consent.

SE-England, United Kingdom.

Ischemic stroke patients of European descent attending a cerebrovascularservice were recruited 1995-2002. All cases were phenotyped by oneexperienced stroke neurologist with review of original imaging (meanage±SD: 64.6±12.7 years, 41% females). Community controls free ofsymptomatic cerebrovascular disease were also recruited by samplingfamily doctor lists from the same geographical region as the patients.Sampling was stratified to provide a similar distribution of age andgender as in the patient group (mean age±SD: 64.8±8.6 years, 41%females). The study was approved by local research ethics committees andinformed consent was obtained from all participants.

Phenotyping.

Only patients with ischemic but not with hemorrhagic strokes wereincluded in the study. All patients had clinically relevant diagnosticwork-up performed, including brain imaging with computed tomography (CT)or/and magnetic resonance imaging (MRI) as well as ancillary diagnosticinvestigations including duplex ultrasonography of the carotid andvertebral arteries, echocardiography, Holter monitoring, MR-angiography,CT-angiography and blood tests. Patients with clinically confirmedTransient Ischemic Attack (TIA) were included in the Ischemic strokegroup from Iceland, Germany-S and Sweden. Patients were classified intoetiologic subtypes according to the Trial of Org 10172 in Acute StrokeTreatment (TOAST)²⁵. This classification includes six categories: (1)large-artery occlusive disease (large vessel disease), (2)cardioembolism (cardiogenic stroke), (3) small vessel disease (lacunarstroke), (4) other determined etiology, (5) etiology unknown despitediagnostic efforts, or (6) more than one etiology. Patients classifiedinto the TOAST categories 4-6 were excluded from the stroke populationfrom Germany-W. In Iceland, patients were classified as havinglarge-artery occlusive disease if stenosis was 70% which is a strictercriterion than usually used i.e. 50%. Classification of stroke patientsinto subtypes according to the Trial of Org 10172 in Acute StrokeTreatment (TOAST) classification system²⁵ in the Icelandic discovery andthe four replication sample sets is listed in Table 1.

IIlumina Genome-Wide Genotyping.

All Icelandic cases and control samples were assayed with the InfiniumHumanHap300 SNP chips (Illumina), containing 317,503 tagging SNPsderived from phase 1 of the International HapMap project. OF the SNPsassayed on the chip, 6,622 SNPs were excluded because they showed either(i) a call rate lower than 95% in cases or controls, (ii) minor allelefrequency less than 1% in the population or (iii) significant distortionfrom Hardy-Weinberg equilibrium in the controls (P<1×10⁻¹⁰). Any samplewith yield<98% were excluded from the analysis. In the final analysis310,881 SNPs were used.

Single SNP Genotyping.

Single-SNP genotyping for all 121 SNP was carried out at deCODE geneticsin Reykjavik, Iceland using the Centaurus (Nanogen) platform²⁶. Thequality of each SNP assay was evaluated by comparing the genotyping ofthe CEU HapMap samples with the publicly available HapMap data. All SNPspassed mismatch tests, linkage disequilibrium (LD) tests and were inHardy-Weinberg equilibrium.

Association Analysis.

For association analysis a standard likelihood ratio statistics wasused, as implemented in the NEMO software created at deCODE²⁷, tocalculate two-sided P values and odds ratio (OR) for each individualallele, assuming a multiplicative model for risk, i.e., that the risk ofthe two alleles a person carries multiply. Allelic frequencies, ratherthan carrier frequencies are presented for the markers.

At the locus on chromosome 4q25, we analysed 3 SNPs, rs2200733,rs10033464 and rs13143308. The third SNP, rs13143308, is in high LD withboth rs2200733 and rs10033464 (D′=0.99 for both) and has a minor allelethat corresponds completely to chromosomes carrying either rs2200733allele T or rs10033464 allele T. It was genotyped in all populationsusing a Centaurus assay, and was used to infer genotypes for thoseindividuals who had missing data for either rs2200733 or rs10033464 onthe Illumina Infinium platform. In Table 21 and Supplementary Table 22,P values and OR for both risk alleles rs2200733-T and rs10033464-T werecomputed on the basis of comparison with the wild-type rs2200733 alleleC, rs13143308 allele G, rs10033464 allele G haplotype, which containsneither of the at-risk alleles¹¹.

For the Icelandic study groups, P values are given after adjustment forthe relatedness of the subjects and other possible populationstratification using the method of genomic control¹⁰. The inflationfactors for the chi-squared statistics are estimated to be 1.07, 1.04,1.06 and 1.02 for the genome-wide association analysis of the IS, CES,LAA of SVD patient groups respectively. With the additional cases andcontrols typed for the 4q locus, we estimated the inflation factorsusing simulations as previously described²⁸. The resulting inflationfactors are 1.09, 1.03, 1.06, 1.05, 1.01, 1.00, 1.01 and 1.00, for thegroups IS, CES, IS excl CES, LVD, SVD, other, unknown and more than onecause, respectively.

Due to the large number of controls used, the effective samples sizeafter adjusting for the relatedness of the cases and controlscorresponds to testing 2,690 IS patients and 2,690 controls. Thecorresponding effective sample sizes for the CES, LAA and SVD patientsare 710, 417 and 467, respectively.

Results from multiple case-control groups were combined using aMantel-Haenszel model in which the groups were allowed to have differentpopulation frequencies for alleles, haplotypes and genotypes but wereassumed to have a common relative risk²⁹

Results

The association of variants within the LD Block C04 region to IschemicStroke was investigated. In order to investigate further thecontribution of the two AF risk variants on 4q25, rs2200733 andrs10033464, to the risk of developing Ischemic Stroke and its subtypes,large artery atherosclerosis (LAA), cardioembolic stroke (CES) and smallvessel disease (SVD), we genotyped marker rs2200733 and markerrs10033464 in Icelandic samples, and for replication purposes we alsoanalyzed replication data sets in cohorts from South-Germany (1,181cases and 1,189 controls, Germany-S), Sweden (1,032 cases and 1,387controls), Westphalia region in Germany (1,388 cases and 1,106 controls,Germany-W), and United Kingdom (654 cases/676 controls, UK). Thephenotype classification of the study cohorts is shown in Table 20.

TABLE 20 TOAST subclassification of genotyped stroke cases, n (%)Replication groups Discovery group United Iceland Germany-S SwedenGermany-W Kingdom Ischemic stroke 1943 1183 1066 1391 654 TOASTsubtyping: 1443 1183 1061 1389 654 Cardioembolism 385 (45) 297 (38) 185(37) 554 (40)  78 (18) Large artery 229 (27) 372 (47) 230 (46) 560 (40)232 (55) atherosclerosis Small vessel disease 246 (29) 118 (15)  82 (16)275 (20) 114 (27) other cause 42 67 56 not recruited 3 more than onecause 34 42 not recruited 40 unknown cause 507 329 466 not recruited 187TOAST = Trial of Org 10172 in Acute Stroke Treatment.

Additional patients (282) and controls (14,893) from Iceland were alsogenotyped for these particular SNPs. The association test was done bycomparing each SNP with the wild-type haplotype (see Methods). As shownin Table 21, rs2200733 conferred an increased risk of Ischemic Stroke inall sample sets, and the association with Ischemic Stroke was highlysignificant with a combined OR=1.26 (P=8.8×10⁻¹¹). For rs10033464, theassociation with Ischemic Stroke was not significant (OR=1.03, P=0.45).Both SNPs however, associated significantly with Cardiembolic Stroke andthis risk was significantly greater than in the Ischemic Stroke group asa whole (rs2200733: OR=1.53, P=1.5×10⁻¹²; rs10033464: OR=1.27,P=5.9×10⁻⁴). This is as expected given the known contribution of AtrialFibrillation to this subphenotype. By removing patients withCardioembolic Stroke from the Ischemic Stroke group, the observed effectfor both SNPs was weaker in the remaining Ischemic Stroke patients, butremained significant for the stronger variant (rs2200733: OR=1.18,P=1.5×10⁻⁵, rs10033464: OR=0.96, P=0.39). Apart from CardioembolicStroke, Large Artery Atherosclerosis and stroke of undetermined causewere the only subphenotypes showing significant association withrs2200733 (OR=1.22, P=1.5×10⁻³, Table 2 and OR=1.18, P=0.01). Theseresults suggest that a significant portion of strokes classified aseither cryptogenic stroke or large artery atherosclerosis may be due toundiagnosed, intermittent AF.

TABLE 21 Association between rs2200733 (allele T) and rs1033464 (alleleT) and Ischemic stroke. rs2200733-T rs10033464-T Phenotype frequencyfrequency Study population (m/n) Controls Cases OR (95% CI) P ControlsCases OR (95% CI) P Ischemic stroke Iceland ( 25708/1943) 0.119 0.1421.23 (1.11-1.36) 4.7 × 10⁻⁵  0.082 0.085 1.07 (0.95-1.21) 0.28 Germany-S(1186/1183) 0.118 0.138 1.19 (1.00-1.41) 0.05 0.093 0.083 0.90(0.73-1.10) 0.31 Germany-W (1107/1391) 0.114 0.146 1.34 (1.13-1.58) 7.0× 10⁻⁴  0.092 0.096 1.10 (0.91-1.33) 0.34 Sweden (740/1066) 0.098 0.1211.27 (1.02-1.58) 0.03 0.113 0.111 1.01 (0.81-1.24) 0.96 UK (676/654)0.087 0.119 1.43 (1.11-1.74) 0.0056 0.090 0.088 1.02 (0.78-1.33) 0.90All groups (29417/6237) 0.107 0.133 1.26 (1.17-1.35) 8.8 × 10⁻¹¹ 0.0940.093 1.03 (0.95-1.12) 0.45 Cardioembolism Iceland ( 25708/385) 0.1190.164 1.50 (1.22-1.85) 1.1 × 10⁻⁴  0.082 0.105 1.39 (1.09-1.79) 0.009Germany-S (1186/297) 0.118 0.175 1.61 (1.25-2.08) 2.5 × 10⁻⁴  0.0930.096 1.11 (0.81-1.52) 0.502 Germany-W (1107/554) 0.114 0.161 1.52(123-1.88) 1.0 × 10⁻⁴  0.092 0.104 1.22 (0.95-1.56) 0.113 Sweden(740/185) 0.098 0.149 1.67 (1.18-2.36) 4.0 × 10⁻³  0.113 0.133 1.28(0.90-1.82) 0.162 UK (676/78) 0.087 0.090 1.08 (0.60-1.95) 0.79 0.0900.122 1.42 (0.83-2.43) 0.198 All groups( 29417/1499) 0.107 0.148 1.53(1.36-1.72) 1.5 × 10⁻¹² 0.094 0.112 1.27 (1.11-1.45) 5.9 × 10⁻⁴ Ischemicstroke excl Cardioembolism Iceland (25708/1558) 0.119 0.136 1.17(1.05-1.31) 0.01 0.082 0.081 1.00 (0.87-1.14) 0.95 Germany-S (1186/886)0.118 0.125 1.06 (0.87-1.28) 0.57 0.093 0.078 0.83 (0.67-1.04) 0.11Germany-W (1107/837) 0.114 0.136 122 (1.01-1.48) 0.04 0.092 0.091 1.02(0.82-1.28) 0.84 Sweden (740/881) 0.098 0.115 1.19 (0.95-1.50) 0.130.113 0.106 0.95 (0.76-1.19) 0.66 UK (676/576) 0.087 0.123 1.48(1.14-1.91) 0.003 0.090 0.083 0.96 (0.73-1.28) 0.80 All groups(29417/4738) 0.107 0.127 1.18 (1.10-1.28) 1.5 × 10⁻⁵  0.094 0.088 0.96(0.88-1.05) 0.39 Large artery atherosclerosis Iceland (25708/229) 0.1190.157 1.41 (1.08-1.86) 0.012 0.082 0.096 1.25 (0.89-1.74) 0.19 Germany-S(1186/372) 0.118 0.117 0.96 (0.75-1.25) 0.78 0.093 0.071 0.74(0.54-1.00) 0.05 Germany-W (1107/560) 0.114 0.140 1.28 (1.03-1.59) 0.030.092 0.100 1.14 (0.89-1.46) 0.30 Sweden (740/230) 0.098 0.094 0.94(0.65-1.34) 0.72 0.113 0.096 0.82 (0.58-1.17) 0.27 UK (676/232) 0.0870.138 1.66 (1.19-2.31) 3.0 × 10⁻³  0.090 0.071 0.82 (0.55-1.23) 0.34 Allgroups (29417/1623) 0.107 0.129 1.22 (1.08-1.38) 1.5 × 10⁻³  0.094 0.0870.96 (0.83-1.11) 0.57 Small vessel disease Iceland (25708/246) 0.1190.112 0.94 (0.71-1.24) 0.64 0.082 0.085 1.03 (0.75-1.42) 0.86 Germany-S(1186/118) 0.118 0.145 1.23 (0.83-1.83) 0.30 0.093 0.063 0.68(0.40-1.14) 0.14 Germany-W (1107/275) 0.114 0.126 1.10 (0.83-1.47) 0.510.092 0.075 0.81 (0.57-1.14) 0.22 Sweden (740/82) 0.098 0.110 1.11(0.66-1.88) 0.70 0.113 0.091 0.80 (0.46-1.37) 0.42 UK (676/114) 0.0870.101 1.18 (0.73-1.91) 0.50 0.090 0.087 0.99 (0.60-1.63) 0.97 All groups(29417/835) 0.107 0.119 1.07 (0.91-1.26) 0.39 0.094 0.080 0.88(0.73-1.05) 0.16 Association results for rs2200733 allele T andrs10033464 allele T for ischemic stroke and the subphenotypes;cardioembolic stroke, large artery atherosclerosis and small vesseldisease, in five study populations. Also presented are the results forischemic stroke after excluding patients with cardioembolism. Resultsfor each phenotype are also included after combining the studypopulations using a Mantel-Haenszel model (All groups). Number ofcontrols (m) and cases (n) is shown in parenthesis, the allelicfrequencies in each group, the OR with a 95% CI and two-sided P valuefor comparison to the wild type haplotype (see Supplementary Methods).The results for the Icelandic population are adjusted for relatedness ofthe individuals.

TABLE 22 rs2200733-T rs10033464-T Phenotype frequency frequency Studypopulation (m/n) Controls Cases OR (95% CI) P Controls Cases OR (95% CI)P Other cause Iceland (25708/42) 0.119 0.155 1.32 (0.72-2.45) 0.37 0.0820.060 0.73 (0.31-1.75) 0.48 Germany-S (1186/67) 0.118 0.119 1.03(0.60-1.77) 0.91 0.093 0.105 1.14 (0.64-2.04) 0.66 Sweden (740/56) 0.0980.125 1.36 (0.74-2.50) 0.32 0.113 0.134 1.26 (0.70-2.26) 0.44 All groups(27634/168) 0.111 0.133 1.19 (0.85-1.66) 0.32 0.096 0.099 1.06(0.74-1.54) 0.74 More than one cause Iceland (25708/34) 0.119 0.088 0.68(0.31-1.52) 0.35 0.082 0.044 0.49 (0.17-1.39) 0.18 Sweden (740/42) 0.0980.112 1.27 (0.61-2.66) 0.52 0.113 0.187 1.84 (0.99-3.41) 0.05 UK(676/40) 0.087 0.213 2.89 (1.54-5.39) 8.9 × 10⁻⁴  0.090 0.088 1.15(0.51-2.61) 0.74 All groups (27124/116) 0.101 0.138 1.48 (0.99-2.21)0.06 0.095 0.106 121 (0.78-1.88) 0.41 Unknown cause Iceland (25708/507)0.119 0.135 1.15 (0.95-1.38) 0.15 0.082 0.073 0.89 (0.70-1.13) 0.35Germany-S (1186/329) 0.118 0.129 1.10 (0.85-1.44) 0.46 0.093 0.087 0.94(0.69-1.28) 0.70 Sweden (740/466) 0.098 0.126 1.32 (1.01-1.71) 0.040.113 0.104 0.94 (0.72-1.23) 0.65 UK (760/187) 0.087 0.102 1.20(0.81-1.78) 0.35 0.090 0.096 1.10 (0.74-1.64) 0.63 All groups(28310/1489) 0.105 0.123 1.18 (1.04-1.34) 0.01 0.095 0.090 0.94(0.82-1.08) 0.41 Association results for rs2200733 allele T andrs10033464 allele T for the TOAST subphenotypes; other cause, more thanone cause and unknown cause in three or four study populations. Resultsfor each phenotype are also included after combining the studypopulations using a Mantel-Haenszel model (All groups). Number ofcontrols (m) and cases (n) is shown in parenthesis, the allelicfrequencies in each group, the OR with a 95% CI and two-sided P valuefor comparison to the wild type haplotype (see Supplementary Methods).The results for the Icelandic population are adjusted for relatedness ofthe individuals.

As discussed in the above (Example 2), the risk alleles of rs2200733 andrs10033464 correlate significantly with the age of diagnosis of AtrialFibrillation. A non-significant trend in the same direction was observedin our study for the age at diagnosis of Cardioembolic Stroke (0.62years per copy of T rs2200733, P=0.33, and 0.29 years per copy of Trs10033464, P=0.71, Table 23), suggesting that the observed age effecton AF may apply to Cardioembolic Stroke also, albeit being a weakereffect.

TABLE 23 Linear regression of age at diagnosis on the number of riskalleles of rs2200733 allele T and rs10033464 allele T. Shown are theregression coefficients and the corresponding two-sided P-valuesobtained using the age at diagnostics as a response (in years) and thenumber of at risk alleles as predictor variables. The sex was includedas a covariate factor in all tests, and also the population in the testfor all groups combined. Numbers of cases used in the analysis are shownin parenthesis (n). rs2200733-T rs2200733-T rs10033464-T rs10033464-Treg. coeff P reg. coeff P Ischemic Iceland (1830) 0.11 0.85 −0.35 0.62Germany-S (1174) 0.29 0.73 0.85 0.43 Sweden (780) 0.56 0.56 1.11 0.23Germany-W (1352) 0.17 0.80 1.21 0.14 UK (654) 0.24 0.83 0.47 0.71 AllGroups (5790) 0.40 0.25 0.68 0.10 Cardioembolic Iceland (356) −1.52 0.16−0.29 0.85 Germany-S (296) −1.72 0.21 −2.53 0.18 Sweden (173) −2.09 0.200.81 0.62 Germany-W (1352) −0.04 1.00 0.82 0.53 UK (78) 7.11 0.084 −5.290.16 All Groups (1441) −0.62 0.33 −0.29 0.71

Discussion

Through this study on 1661 Icelandic IS patients and 10815 controls andthe follow-up replication in large and well characterized EuropeanIschemic Stroke case/control sample sets we identified and validated arisk variant on chromosome 4q25, tagged by rs2200733, that associateswith Iscemic Stroke. In our study, as expected, these variantsassociated most strongly with the subphenotype Cardioembolic Stroke,which is a major complication of Atrial Fibrillation. The risk that isobserved in Ischemic Stroke patients without Cardioembolic Stroke ispossibly due to an underdiagnosis of Atrial Fibrillation and therebyCardioembolic Stroke, since Atrial Fibrillation is often asymptomatic orintermittent and can consequently be difficult to detect in strokepatients.

Up to 30% of Ischemic Stroke are caused by cardioembolism(5, 6) of whicha large proportion occurs in the presence of Atrial Fibrillation(7, 8).Atrial Fibrillation is the most common sustained cardiac arrhythmia ofman and its prevalence increases with age, affecting approximately 10%of those over 80 years of age (3, 9). As such, AF is one of the mostpowerful independent risk factors for stroke and on a population level,AF is associated with a fourfold to fivefold increase in the risk ofstroke(3, 7, 8, 10). Moreover, Caridembolic Stroke is generally severe,reflected by greater disability, higher rates of stroke recurrence andhigher mortality than in other subtypes of strokes (6, 11). Earlydetection of those at risk for AF is important in order to reduce therisk of suffering a future stroke. Clinical trials on stroke preventionin patients with AF have shown that anticoagulant medications (e.g.warfarin) reduce the risk of stroke substantially(7, 12) and is muchmore effective than anti-platelet agents such as aspirin andclopidogrel. Our results strongly suggest that a significant portion ofstroke patients have undiagnosed atrial fibrillation and are classifiedeither as cryptogenic stroke or as large vessel stroke. Such patientsmay have asymptomatic, intermittent AF that is not detected duringroutine workup of 24 to 48 hours of cardiac monitoring. This issupported by two studies of post-stroke patients who underwent another 4to 7 days of ambulatory cardiac monitoring; the rates of intermittent AFpreviously undiagnosed were 5.6 and 14.3% (13, 14). Stroke patients withasymptomatic or intermittent AF would be inadequately treated ifmisdiagnosed instead as e.g. cryptogenic stroke or large vessel strokesince such patients are placed on an anti-platelet agent instead ofwarfarin. Therefore, these markers for AF may help determine whichpatient might benefit from prolonged cardiac monitoring as an outpatientto document the presence or absence of AF. Prospective studies areneeded to determine whether these findings can be translated into betterprevention or treatment for stroke.

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1. A method of determining a susceptibility to cardiac arrhythmia orstroke in a human individual, the method comprising determining thepresence or absence of at least one allele of at least one polymorphicmarker in a nucleic acid sample from the individual, wherein the atleast one polymorphic marker is selected from the polymorphic markersset forth in Table 5, and markers in linkage disequilibrium therewith,and wherein determination of the presence or absence of the at least oneallele is indicative of a susceptibility to cardiac arrhythmia or strokein the individual.
 2. (canceled)
 3. The method of claim 1, wherein theat least one polymorphic marker is selected from the markers set forthin Table 9, and markers in linkage disequilibrium therewith.
 4. Themethod of claim 1, wherein the at least one polymorphic marker isselected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733(SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), andrs13143308 (SEQ ID NO:51), and markers in linkage disequilibriumtherewith.
 5. The method of claim 1, wherein the presence of alleles-2,-4 and/or -8 in marker D4S406, allele A of marker rs2634073, allele T ofmarker rs2200733, allele T of marker rs2220427, allele T of markerrs10033464, and/or allele G of marker rs13143308 is indicative ofincreased susceptibility of cardiac arrhythmia or stroke in theindividual.
 6. The method of claim 1, wherein the susceptibility isincreased susceptibility characterized by an odds ratio (OR) of at least1.3. 7-25. (canceled)
 26. A method of determining a susceptibility tocardiac arrhythmia or stroke in a human individual, the methodcomprising determining the identity of at least one allele of at leastone polymorphic marker in a nucleic acid sample obtained from theindividual, wherein the at least one marker is selected from the groupof markers associated with the PITX2 gene, wherein the presence of theat least one allele is indicative of a susceptibility to cardiacarrhythmia or stroke in the individual.
 27. The method according toclaim 26, wherein the at least one marker is selected from rs7668322(SEQ ID NO:46), rs2197815 (SEQ ID NO:47), rs6831623 (SEQ ID NO:48) andrs259110 (SEQ ID NO:49), and markers in linkage disequilibriumtherewith.
 28. The method of claim 26, wherein the susceptibility isincreased susceptibility.
 29. (canceled)
 30. The method of claim 1,wherein the cardiac arrhythmia is atrial fibrillation or atrial flutter.31-33. (canceled)
 34. The method of claim 1, wherein the stroke isischemic stroke.
 35. The method of claim 1, wherein linkagedisequilibrium is characterized by numerical values for r² of greaterthan 0.2. 36-48. (canceled)
 49. The method of claim 1, furthercomprising assessing at least one biomarker for atrial fibrillation,atrial flutter and/or stroke in a sample from the individual.
 50. Themethod of claim 1, further comprising analyzing non-genetic informationto make risk assessment, diagnosis, or prognosis of the individual. 51.The method of claim 50, wherein the non-genetic information is selectedfrom age, age at onset of disease, gender, ethnicity, socioeconomicstatus, previous disease diagnosis, medical history of subject, familyhistory of atrial fibrillation, atrial flutter and/or stroke,biochemical measurements, and clinical measurements.
 52. The method ofclaim 49, further comprising calculating overall risk by logisticregression. 53-74. (canceled)
 75. An apparatus for determining a geneticindicator for cardiac arrhythmia and/or stroke in a human individual,comprising: a computer readable memory; and a routine stored on thecomputer readable memory; wherein the routine is adapted to be executedon a processor to analyze genotype and/or haplotype data for at leastone human individual with respect to at least one polymorphic markerselected from the markers set forth in Table 5, and markers in linkagedisequilibrium therewith, and generate an output based on the marker orhaplotype data, wherein the output comprises a risk measure of the atleast one marker or haplotype as a genetic indicator of cardiacarrhythmia and/or stroke for the human individual.
 76. The apparatus ofclaim 75, wherein the routine further comprises determining an indicatorof the frequency of at least one allele of at least one polymorphicmarker and/or at least one haplotype in a plurality of individualsdiagnosed with cardiac arrhythmia and/or stroke, and an indicator of thefrequency of at the least one allele of at least one polymorphic markeror at least one haplotype in a plurality of reference individuals, andcalculating a risk measure for the at least one allele and/or haplotypebased thereupon; and wherein a risk measure for the individual iscalculated based on a comparison of the at least one marker and/orhaplotype status for the individual to the calculated risk for the atleast one marker and/or haplotype information for the plurality ofindividuals diagnosed with atrial fibrillation, atrial flutter and/orstroke.
 77. The apparatus of claim 75, wherein the at least onepolymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073(SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1),rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51).
 78. Theapparatus of claim 75, wherein the risk measure is characterized by anOdds Ratio (OR) or a Relative Risk (RR).
 79. The apparatus or medium ofclaim 75, wherein linkage disequilibrium is characterized by values ofr² of greater than 0.2.